Inhibition of siderophore biosynthesis in Mycobacterium tuberculosis with nucleoside bisubstrate analogues: Structure-activity relationships of the nucleobase domain of 5′-O-[N-(salicyl)sulfamoyl]adenosine

J. Med. Chem. 2008, 51, 5349–5370

5349

Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside

Bisubstrate Analogues: Structure-Activity Relationships of the Nucleobase Domain of

5′-O-[N-(Salicyl)sulfamoyl]adenosine

Joa˜o Neres,^†Nicholas P. Labello,^†Ravindranadh V. Somu,^†Helena I. Boshoff,^‡Daniel J. Wilson,^†Jagadeshwar Vannada,^†

Liqiang Chen,^†Clifton E. Barry III,^‡Eric M. Bennett,^†and Courtney C. Aldrich*^,†

Center for Drug Design, Academic Health Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Tuberculosis Research

Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892

ReceiVed May 14, 2008

5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is a prototype for a new class of antitubercular agents that

inhibit the aryl acid adenylating enzyme (AAAE) known as MbtA involved in biosynthesis of the mycobactins.

Herein, we report the structure-based design, synthesis, biochemical, and biological evaluation of a

comprehensive and systematic series of analogues, exploring the structure-activity relationship of the purine

nucleobase domain of Sal-AMS. Signiﬁcantly, 2-phenyl-Sal-AMS derivative 26 exhibited exceptionally potent

antitubercular activity with an MIC₉₉under iron-deﬁcient conditions of 0.049 µM while the N-6-cyclopropyl-

Sal-AMS 16 led to improved potency and to a 64-enhancement in activity under iron-deﬁcient conditions

relative to iron-replete conditions, a phenotype concordant with the designed mechanism of action. The

most potent MbtA inhibitors disclosed here display in vitro antitubercular activity superior to most current

ﬁrst line TB drugs, and these compounds are also expected to be useful against a wide range of pathogens

that require aryl-capped siderphores for virulence.

Introduction

for antibiotic development.⁴This approach is particularly

Tuberculosis (TB^a) is the leading cause of infectious disease

mortality by a bacterial pathogen responsible for over 1.5 million

deaths each year.¹While the course of treatment may vary, the

shortest recommended therapy for active infection involves no

less than 182 doses of a multifrontline antibiotic cocktail over

a 6 month period.²Increasing reports of drug resistant Myco-

bacterium tuberculosis strains, namely, the virtually incurable

extensively drug-resistant TB (XDR-TB) and the serious mul-

tidrug resistant TB (MDR-TB), are cause of great concern, as

they are resistant to both ﬁrst-line and second-line anti-TB

drugs.³Additionally, co-infection with HIV/AIDS is now

responsible for the majority of HIV-related deaths. New TB

drugs with shortened periods of treatment that are effective

against latent TB infections are urgently needed.

Free iron is an essential nutrient for almost all known

organisms, but its concentration in serum and human body ﬂuids

is approximately 10^-24M, far too low to support bacterial

colonization and growth. Many bacterial pathogens secrete and

reimport small molecule iron-chelators termed siderophores for

iron acquisition, making siderophore biosynthesis a logical target

attractive with the discovery that many antibiotics ultimately

disrupt iron homeostasis.⁵Iron has also recently been shown to

play a key role in bioﬁlm formation in Mycobacterium smeg-

matis.⁶Mutational analysis demonstrated that siderophore

synthesis was required for bioﬁlm formation.

In response to iron starvation, M. tuberculosis produces two

series of structurally related siderophores that vary in the

appended lipid side chain collectively referred to as mycobactins,

critical for virulence and growth (4 and 5, Figure 1).^7,8

Mycobactins are required for virulence in THP-1 macrophages

and in a murine model of TB infection and may also serve as

a short-term iron reservoir potentially mediating reactive oxygen

species (ROS) generation.^7–10Biosynthesis of these aryl-capped

siderophores is initiated by the aryl acid adenylation enzyme

MbtA (Figure 1), which activates salicylic acid (1) forming an

acyladenylate intermediate (Sal-AMP, 2). MbtA is also respon-

sible for loading the acyladenylate intermediate onto the

thiolation domain of MbtB.¹¹The remaining steps of myco-

bactins biosynthesis are catalyzed by a mixed nonribosomal

peptide synthetase polyketide synthase (NRPS-PKS) assembly

line.¹¹

Recently, 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS, 6,

Figure 2), a rationally designed bisubstrate inhibitor, was

reported as a stable mimic of the intermediate acyladenylate

2.^12–14Developing this compound as a useful probe and a

potential antibacterial agent requires improving target speciﬁcity,

potency, and lipophilicity. Herein, we report the structure-based

design of a potent picomolar inhibitor with enhanced speciﬁcity

and in vitro antitubercular activity superior to most current ﬁrst

line TB drugs.

* To whom correspondence should be addressed. Phone: 612-625-7956.

Fax: 612-626-5173. E-mail: aldri015@umn.edu.

†

University of Minnesota.

National Institute of Allergy and Infectious Diseases.

‡

^aAbbreviations: AAAE, aryl acid adenylating enzyme; AMP, adenosine

monophosphate; ATP, adenosine triphosphate; DMA, dimethylacetamide;

DTT, dithiothreitol; HIV/AIDS, human immunodeﬁciency virus/acquired

immune deﬁciency syndrome; HMWP2, high-molecular-weight protein 2;

K_i^app, apparent inhibition constant; MDR-TB, multidrug resistant tubercu-

losis; MIC, minimum inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide; NHS, N-hydroxysuccinimide; NRPS,

nonribosomal peptide synthetase; PDB, Protein Data Bank; PKS, polyketide

synthase; PP_i, pyrophosphate; QM/MM, quantum mechanical/molecular

mechanical; rmsd, root-mean-square deviation; ROS, reactive oxygen

species; SAR, structure activity relationships; Sal-AMS, 5′-O-[N-(salicyl-

)sulfamoyl]adenosine; TB, tuberculosis; TBAF, tetrabutylammonium ﬂuo-

ride; TFA, triﬂuoroacetic acid; XDR-TB, extensively drug resistant

tuberculosis.

Results

Molecular Modeling. A complete homology model of MbtA

was constructed on the basis of the cocrystal structure of DhbE

with an adenylated 2,3-dihydroxybenzoic acid (see Experimental

10.1021/jm800567v CCC: $40.75

2008 American Chemical Society

Published on Web 08/09/2008

5350 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

Figure 1. Biosynthesis of the mycobactins and carboxymycobactins.⁸The depicted lipid side chain is representative, as both 4 and 5 are isolated

as mixtures with various length lipid residues.

Figure 3. Compound 16 (colored by element, hydrogens not shown)

is superimposed on a molecular surface representation of MbtA. The

binding pocket and N6 cleft are clearly visible. Substituents larger than

three carbons extend beyond the available volume and conﬂict sterically

Figure 2. Bisubstrate inhibitors of MbtA. The expanded portion of

the ﬁgure shows the nucleobase modiﬁcations described herein.

with the protein.

binding pocket into volume occupied by the protein. The N-6

pocket is clearly limited to no more than three carbon atoms

for optimal potency in agreement with the small available

volume observed in the MbtA homology model.

Compounds 20 and 21 showed unfavorable ligand-ligand

van der Waals contacts between the C-8 substituent and the

salicyl moiety in the compact receptor-bound conformation

demonstrated in Figures 3 and 4A. Tan and co-workers recently

reported an innovative approach to improving selectivity for

related aminoacyl adenylate inhibitors by introduction of a

conformation constraint through modiﬁcation at C-8.¹⁶However,

our results suggest this strategy will be more challenging to

implement for Sal-AMS.

Substitution at C-2 was initially explored with 2-iodo 23,

2-phenylamino 24, phenylethynyl 25, and 2-phenyl 26 in order

to occupy the additional space at this binding site predicted by

molecular dynamics simulations. QM/MM studies on compound

26 predicted the binding conformation shown in Figure 4A, with

the C-2 phenyl group positioned between the side chain of

Lys332 and the backbone atoms of Val448. The original

conformations of these two residues (in green) blocked access

to this pocket, whereas the alternative conformation provides

enough room for C-2 substituents such as the phenyl group in

26 or even larger groups. In particular, further space was still

available in this region at either the meta or para positions of

the phenyl ring in 26; thus, a series of biphenyl derivatives

27-29 were evaluated (see Figure S5, Supporting Information).

QM/MM studies on compounds 27-29 showed that substitution

at the meta and para positions in m-biphenyl 28 and p-biphenyl

29 successfully access the interdomain pocket (Figure 4B).

Substitution at the ortho position in o-biphenyl 27 crowds the

N-3 pocket rather than exploiting the interdomain region in the

Section). The active sites are 76% identical, and both show a

small unexploited pocket vicinal to N-6 (purine moiety of 6),

as well as a larger cavity near N-3 adjacent to the linker that

connects the N- and C-terminal domains.¹⁵Additionally, a 30

ns molecular dynamics simulation of MbtA showed a hydrogen

bond between the Lys332 ꢀ-amino and Val448 backbone

carbonyl that repeatedly breaks and re-forms, increasing the

space available near C-2 when broken (see Figures S1-S4,

Supporting Information). Finally, the purine/protein interactions

are largely hydrophobic, with no major electrostatic interactions

present for N-1 and N-3 and only poorly aligned hydrogen bonds

between N-7 and the NH of Gly330 and Gly354. On the basis

of this analysis, a comprehensive series of 23 compounds (7-29,

Figure 2) were designed to explore the importance of the purine

N-1, N-3, and N-7 atoms, the role of the exocyclic N-6 amino

group, and the impact of substitution at C-2 and C-8. These

structures were docked and analyzed within the MbtA homology

model.

In the N-6 alkylamino analogues series, docking of com-

pounds 11-14 resulted in purine heavy atom rmsd values of

less than 0.8 Å relative to 6. Compound 15 displaces these atoms

over 1.8 Å, indicating that the isobutyl substituent is too large.

Similarly, the cyclic analogues 16 and 17 displace the base 0.5

and 0.7 Å, respectively, while the bulkier cyclopentyl and benzyl

groups in 18 and 19 result in an rmsd of 1.4 Å. Accordingly,

compounds 11-14 and 16-17 retain the hydrogen bond from

the purine N-6 to the carbonyl of Val352 originally observed

for 6 (see Figure 4A) while compounds 9 and 10 are unable to

perform this hydrogen bond. Figure 3 presents 16 superimposed

on a surface representation of MbtA. The cyclopropyl group

ﬁlls the N-6 cleft, while larger substituents extend beyond the

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5351

Scheme 1^a

^aReaction conditions: (a) 2,2-dimethoxypropane, CSA, acetone, 88%;

(b) NH₂SO₂Cl, NaH, DME, 79%; (c) 33, Cs₂CO₃, DMF; (d) 80% aqueous

TFA, 41% over two steps.

Scheme 2^a

Figure 4. (A) Model of subpicomolar inhibitor 26 bound to MbtA in

the predicted binding mode (QM/MM/6-31G(d)). The protein is reduced

to a ribbon diagram with the N-terminal in red and C-terminal in blue.

Lys332, Val352, and Val448 are shown as sticks (color by atom). The

conformations of Lys332 and Val448 in the original homology model

with compound 6 are shown in green, indicating the conformational

shift necessary to permit binding of ligands with large C2-substituents.

The hydrogen bond from N-6 to Val352 explains the ∼238-fold activity

difference between the N-6-dimethyl 10 and N-6-methyl 11 analogues.

Distances are given in angstroms. (B) Model of m-biphenyl 28 bound

to MbtA in the predicted binding mode (QM/MM/6-31G(d)). The

protein is reduced to a ribbon diagram with the N-terminal in red

(residues 1-443), C-terminal in blue (residues 455-558), and linker

in gray (residues 444-454) to demonstrate the location of C2-

substituents with respect to the tertiary structure of the protein.

^aReaction conditions: (a) NaH, THF, 30 min, then PhSO₂Cl, 1.5 h, 62%;

(b) NaBH₄, MeOH, 0 °C, 1.5 h, 100%; (c) NaH, THF, 30 min, then pivaloyl

chloride, reﬂux, 4 h, 88%; (d) 4-nitroindole, NaH, Pd₂(dba)₃·CHCl₃(0.025

equiv), P(O-i-Pr)₃(0.20 equiv), 3 h, 55%; (e) OsO₄(0.05 equiv), NMO

(2.0 equiv), THF, 2.5 h; (f) p-TsOH, 2,2-dimethoxypropane, 24 h, 50%

(∼1:1, 39/40) over two steps; (g) NaOH, H₂O/MeOH, 100 °C, 2 h, 89%;

(h) NH₂SO₂Cl, DMA, 0 °C, 2.5 h, 86%; (i) 33, Cs₂CO₃, DMF, 16 h, 77%;

(j) p-TsOH (1.2 equiv), MeOH, 70 °C, 3 h, 46%; (k) Pd/C, H₂(1 atm),

DMF, 8 h, 44%.

problematic owing to poor anomeric control compounded by

difﬁculty in anomer separation.²⁰Therefore, we elected to

replace the ribofuranosyl ring oxygen with carbon to circumvent

these complications, a modiﬁcation that we previously showed

was well tolerated.²¹Installation of the indole moiety was

accomplished via a palladium catalyzed allylic amination of 37

with 4-nitroindole that proceeded with excellent regio- and

stereocontrol via a π-allyl intermediate to afford 38 in 55% yield

as the only isolated coupled product (Scheme 2).²²This

innovative reaction developed by Jung and Rhee represents a

powerful and efﬁcient method for the preparation of carbocyclic

nucleoside analogues. Cyclopentylamine substrate 37 was

derived from Vince lactam 34²³in three steps. Utilization of

the phenylsulfonyl group over the reported tosyl group in this

manner of the other C-2 modiﬁcations. Steric clashes with the

protein and quite possibly the purine ring indicate that large

substituents in this position will result in increasingly strained

conformations of the ligand and reduced potency.

Chemistry. The synthesis of 7-deaza analogue 7 commenced

from tubercidin 30, a nucleoside antibiotic produced by Strep-

tomyces tubercidus¹⁷(Scheme 1). Protection of 30 as the

acetonide followed by sulfamoylation afforded 32.¹⁸Salicylation

with NHS ester 33 mediated by Cs₂CO₃and subsequent

deprotection with 80% aqueous TFA furnished 7.^14,19

The importance of N-1 and N-3 of the purine moiety was

explored with the preparation of indole analogue 8. The

synthesis of indole nucleosides in general is low yielding and

5352 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

Scheme 3^a

Scheme 4^a

^aReaction conditions: (a) 2,2′-dimethoxypropane, CSA, acetone, 65%;

(b) NaN₃, DMF, 70 °C, 12 h, 83%; (c) NH₂SO₂Cl, NaH, DME, 48-85%;

(d) 33, Cs₂CO₃, DMF; (e) 80% aqueous TFA, 55-57% over two steps; (f)

Pd/C, H₂(1 atm), MeOH, 2 h, 73%.

Scheme 5^a

^aReaction conditions: (a) three steps, 80% overall yield;;²⁸(b) 2,2′-

dimethoxypropane, CSA, acetone, 76%; (c) NH₂SO₂Cl, NaH, DME, 92%;

(d) 33, Cs₂CO₃, DMF; (e) 80% aqueous TFA, 50% over two steps; (f) RNH₂

(1.5 equiv), Et₃N (3.0 equiv), EtOH, 75 °C, 5 h, sealed tube; (g)

2,2-dimethoxypropane (2.0 equiv), p-TsOH (1.0 equiv), acetone, 16 h,

49-96%, two steps; (h) NH₂SO₂Cl, NaH, DME, 67-91%; (i) 33, Cs₂CO₃,

DMF, 39-85%; (j) 80% aqueous TFA; (k) Dowex 50WX2-Na⁺, 20-54%,

two steps.

sequence (34 f 37) enabled improved overall yields (55% vs

42%).^22,24Dihydroxylation of 38 followed by acetonide protec-

tion provided a separable mixture of diasteromeric carbocyclic

nucleosides 39 and 40 in an approximately 1:1 ratio.²⁵The

relative stereochemistry was conﬁrmed by NOE studies. Sul-

famoylation of 39 with sulfamoyl chloride in DMA according

to the Okada protocol²⁶followed by salicylation with 33

provided 43. Attempted acetonide deprotection employing 80%

aqueous TFA afforded a deep-purple solution and concomitant

decomposition likely due to acid catalyzed indole dimerization.

Milder deprotection conditions with p-TsOH in MeOH suc-

cessfully provided 44. The electron withdrawing nitro group

served to deactivate the indole moiety during the acid mediated

acetonide deprotection and dictated the order of the last two

reaction steps. Finally, hydrogenation of the nitro moiety of 44

with Pd/C in EtOAc provided 8, which was isolated as the

triethylammonium salt. Notably, hydrogenation of 44 in MeOH

led to major byproducts arising from mono- and dimethylation

of the amino group.²⁷

Synthesis of N-6 substituted derivatives was achieved via a

common reaction sequence (Scheme 3). The key starting

material was prepared from inosine in three steps by sequential

triﬂuoroacetylation, chlorination, and methanolysis as described

to provide 48.²⁸Amination of 48 was performed with 3.0 equiv

of triethylamine in EtOH in a sealed tube at 70 °C.²⁹For very

low molecular weight amines (methylamine, ethylamine, cy-

clopropylamine) solvolysis without Et₃N was preferred. The

crude nucleosides were directly converted to the corresponding

acetonides by treatment with p-TsOH and 2,2-dimethoxpropane

in acetone to afford nucleosides 49-58. Sulfamoylation

provided 59-68 and subsequent salicylation with 33 followed

by acetonide deprotection with 80% aqueous TFA furnished

^aReaction conditions: (a) ref 32; (b) PdCl₂(PPh₃)₂(0.1 equiv), CuI (0.2

equiv), Et₃N (2.1 equiv), DMF, 80 °C, 16 h, 57%; (c) ref 32; (d) 2-, 3-, or

4-biphenylboronic acid (2.0 equiv), Pd(OAc)₂(0.2 equiv), 2-(biphenyl)di-

cyclohexylphosphine (0.3 equiv), K₃PO₄(2.5 equiv), 100 °C, 12 h, 58-68%;

(e) NH₂SO₂Cl, NaH, DME, 37-90%; (f) NH₂SO₂Cl, DMA, 0 °C, 3 h,

96-97%; (g) 33, Cs₂CO₃, DMF; (h) 80% aqueous TFA, 45-72% over

two steps.

10-19 isolated as the triethylammonium salts that were

converted to the sodium salts by ion exchange with a strong

cation exchange resin in the sodium form. Derivative 9 was

prepared from inosine in four steps in analogy to the

preparation of 7 (Scheme 3).

The impact of substitution at C-8 of the purine moiety was

assessed with the preparation of 20-22 (Scheme 4). 8-Bro-

moadenosine 69 was protected as acetonide 70, which was

converted to 8-azido 71 by treatment with NaN₃in DMF

(Scheme 4).³⁰Both 70 and 71 were elaborated to the corre-

sponding sulfamates 72 and 73 and then salicylated with 33

and deprotected to provide 20 and 21 in analogy to the

preparation of 7. Hydrogenation of 21 afforded 8-amino

derivative 22.

Modiﬁcation of C-2 was most efﬁciently achieved from

2-iodoadenosine, which enabled installation of various substit-

uents via standard palladium catalyzed reactions as described

below (Scheme 5). 2-Iodo-2,3-O-isopropylieneadenosine 75 was

prepared in ﬁve steps from guanosine following the Matsuda

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5353

Table 1. Inhibition of MbtA and Whole Cell M. tuberculosis H37Rv

^aZ ) O for compounds 6, 7, 9-29; Z ) CH₂for compound 8. ^bAssay performed with 7 nM MbtA, 10 mM ATP, 250 µM salicylic acid, 1 mM PP_i.

^cGrown in normal pH 6.6 glycerol-alanine-salts (GAS) medium without ferric ammonium citrate. ^dGrown in normal pH 6.6 glycerol-alanine-salts

(GAS) medium supplemented with 200 µM ferric ammonium citrate. ^en.d.: not determined. ^fN-1 of the purine is protonated.

protocol.³¹Buchwald-Hartwig coupling of 75 with aniline

afforded the phenylamino derivative 76.³²Similarly, Sonogash-

ira coupling of 75 with phenylacetylene furnished 77 and Suzuki

coupling of 75 with phenylboronic acid as well as 2-, 3-, and

4-biphenylboronic acid provided 78-81, respectively.³²The

palladium-mediated reactions required a 20 mol % catalyst

loading in order to obtain complete conversion. We speculate

the moderate yields observed in these palladium-catalyzed

reactions is likely due to coordination of Pd(II) to N¹and N⁷

of the purine.³³Such bidentate complexes are expected to have

lower catalytic activity and may contribute to deglycosylation

of the nucleoside. Elaboration of nucleosides 75-81 to sulfa-

mates 82-88 and salicylation and subsequent deprotection to

23-29 were carried out in analogy to the preparation of 7.

MbtA Inhibition. Inhibition of MbtA was performed using

an ATP-PP_iexchange assay as previously described²¹(see

Experimental Section). Assays were performed at 37 °C with

recombinant MbtA expressed in E. coli in a buffer of 75 mM

Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM DTT, 250 µM salicylic

acid, 10 mM ATP, and 1 mM PP_i.²¹The initial rates of

pyrophosphate exchange (e10% reaction) were monitored by

measuring the amount of [³²P]ATP formed after addition of

[³²P]PP_i. The apparent inhibition constants (K_i^app) were deter-

mined by ﬁtting the concentration-response plots either to the

Hill equation (eq 1; see Experimental Section) or to the Morrison

equation (eq 2; see Experimental Section). The Morrison

equation was employed in cases where the inhibitors exhibited

tight-binding behavior (K_i^appe 100 × [E]).

The K_i^appof the parent compound 6 has been previously

determined to be 6.6 nM (Table 1).²¹The impact of N-7 was

evaluated with 7-deaza-analogue 7, which resulted in a 4-fold

loss of potency, consistent with loss of the hydrogen bonds

between N-7 and the NH of Gly330 and Gly354 (Table 1).

Removal of N-1 and N-3 in indole derivative 8 demonstrated

that these nitrogens are dispensable for activity.

Inosine derivative 9 and dimethylamino 10 led to a 121-fold

and 58-fold loss of potencies respectively, establishing the

requirement for at least one hydrogen bond donor appended at

C-6. Therefore, a systematic series of monoalkyl and cycloalkyl

N-substituted analogues 11-19 were synthesized revealing that

small C1-C3 alkyl groups were well tolerated, as predicted by

the homology model, which showed a small hydrophobic pocket

in the protein structure vicinal to N-6. Cyclopropyl 16 was the

most potent analogue in this series with a 3.5-fold enhancement

in binding afﬁnity. The potency rapidly diminished as the alkyl

chain increased beyond C-3; thus, cyclobutyl 17 and isobutyl

15 exhibited a 124- and 1140-fold loss in potencies, respectively.

Further increases in chain length with 18 and 19 led to an

approximately 10,000-fold decrease in binding afﬁnity.

5354 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

Inhibitors 20-22 bearing substitution at C-8 resulted in 300-

to 30,000-fold loss potency concordant with predictions from

molecular modeling. Additionally, the propensity of bulky

groups at C-8 of adenine to favor the syn conformation about

the glycosidic linkage could account for part of the reduced

activity, since the inhibitor must bind in an anti conformation

to MbtA.³⁴

Substitution at C-2 was initially explored with 2-iodo 23,

which exhibited a 2-fold increase in afﬁnity. Encouraged by

this result, a small series of analogues were synthesized by

various cross-coupling reactions to provide 2-phenylamino 24,

phenylethynyl 25, and 2-phenyl 26. These compounds were 7,

17, and 24 times more potent than 6. Modeling studies predicted

that additional room at this binding site was available at either

the meta or para positions of the phenyl ring in 26. Accordingly

biphenyl derivatives 27-29 were prepared and found to exhibit

the following trend: meta ∼ para > ortho. The 174-fold loss in

potency of 27 compared to 26 is consistent with a steric clash

of the biphenyl with backbone residues in the N-terminal

domain, while the meta and para phenyl groups successfully

access the interdomain pocket. The ability to tolerate these bulky

nonpolar substituents at C-2 was remarkable but consistent with

our molecular dynamics simulations, which showed a high

degree of ﬂexibility in this region.

Cell Cytotoxicity. To assess potential toxicity, all of the

compounds were evaluated against Vero cells using an MTT

assay and found not to inhibit cell growth (IC₅₀> 100 µM),

providing a therapeutic index (IC₅₀/MIC₉₉) greater than 2000

for 24-26.

Mechanism of Action Studies. In an effort to delineate the

putative secondary mechanism of action of these adenylation

inhibitors, 6 was evaluated for enzyme inhibition against a panel

of adenylating enzymes (FadD17, FadD19, FadD26, and

FadD28) involved in fatty acid metabolism in M. tuberculosis

(see Experimental Section).³⁶However, 6 showed no inhibition

of any of the four FadDs evaluated at 100 µM. MbtA is

functionally related to aminoacyl t-RNA synthetases involved

in protein synthesis. Therefore, 6 and 5′-O-(sulfamoyl)adenos-

ine,³⁷a potent inhibitor of protein synthesis, were evaluated

for inhibition of protein synthesis using an in vitro protein

translation assay (see Experimental Section).³⁷Remarkably, 6

exhibited no protein inhibition (IC₅₀> 100 µM) despite the

structural similarity to 5′-O-(sulfamoyl)adenosine.

Discussion

Given the importance of mycobacterial iron metabolism and

the critical function of the mycobactins, the design of small

molecule siderophore biosynthesis inhibitors represents a novel,

promising strategy to develop new antitubercular agents.^4,12–14,21

An alternative approach developed by Miller and co-workers

involves the synthesis of siderophore analogues as potential

antagonist of siderophore function. Indeed a full-length myco-

bactin analogue was found to have potent activity with an MIC

of 0.2 µg/mL.³⁸Quadri and co-workers recently identiﬁed a

series of thiacarbamoylpyrazoline analogues designed as small

molecule mimics of the terminal salicyl-oxazoline cap of

mycobactin with several compounds displaying low micromolar

antitubercular activity and selectivity under iron deﬁcient

conditions.³⁹Finally, it is noted that a conceptually related and

very successful approach also pioneered by Miller and co-

workers involves the synthesis of siderophore-antibiotic con-

jugates that exploit siderophore receptors for uptake and delivery

of the antibiotics.⁴⁰

Isothermal Calorimetry. The most potent ligand, 2-phenyl-

Sal-AMS 26, was further characterized and shown to be an

enthalpy-driven reversible competitive inhibitor with respect to

salicylic acid and ATP. Notably, the reported K_i^appvalues vastly

underestimate potency of these bisubstrate inhibitors, since the

K_i^appvalues were determined under supersaturating concentra-

(Sal)

tions of both substrates salicylic acid (250 µM or 120K_M

)

and ATP (10 mM or 55K_M^(ATP)). Attempts to determine its true

inhibition constant (K_i) were complicated by the tight-binding

behavior and the bisubstrate nature of inhibition, which pre-

cluded K_iassessment by traditional steady state kinetic meth-

ods.³⁵Direct measurement of the dissociation constant (K_D) was

obtained by isothermal calorimetry in the presence of saturating

salicylic acid at 298 K, which afforded K_D, ∆H, and -T∆S

values of 0.26 ( 0.23 pM, -15.9 ( 0.6 kcal/mol, and -1.6 (

1.4 kcal/mol, respectively.

In this report, we have used molecular modeling to guide

modiﬁcations to the nucleobase domain of Sal-AMS 6. Deletion

of N-1 or N-3 in the purine ring was tolerated, while removal

of N-7 led to a slight decrease in inhibitor potency. We found

that at least one hydrogen bond donor at N-6 is essential for

activity of this class of compounds, as inosine 9 and dimethyl

10 analogues exhibited pronounced reductions in potency.

Substitution of N-6 with C1-C3 chains was beneﬁcial leading

to incremental increases in potency reaching a maximum with

cyclopropyl 16. The potency followed a logarithmic decline as

the chain length increased to C4 and C5, then leveled off at

C5. Although compounds 11-13 exhibited the same antituber-

cular activity as the parent compound 6, the N-6 cyclopropyl

analogue 16 showed a signiﬁcant improvement in biological

activity. Additionally, 16 displayed the best selectivity of any

Sal-AMS derivative investigated with a 64-fold difference in

antitubercular activity between iron-deﬁcient and iron-rich

conditions. The C-8 modiﬁed analogues 20-22 exhibited

drastically reduced potency, illustrating the exquisite sensitivity

to modiﬁcation at this position that had been predicted by our

homology model. The most potent compounds, both in the in

vitro and the whole-cell assays, were in the C-2 modiﬁed series,

with MbtA showing a remarkable ability to tolerate bulky

nonpolar substituents in this position. By contrast, relatively

modest changes to the salicyl, sulfamoyl, and ribosyl subunits

Antitubercular Activity. The antitubercular activity of these

compounds was evaluated against whole-cell M. tuberculosis

H37Rv under iron-deﬁcient and iron-replete conditions. The

whole cell assay permits the assessment of M. tuberculosis

inherent resistance because of factors such as limited membrane

permeability. Results are given in Table 1, in the form of MIC₉₉,

the minimum concentration of compound that completely

inhibited observable growth of M. tuberculosis H37Rv.

Overall the MIC₉₉values are well correlated with the K_i^app

values and analogues 24-26 exhibited the most potent whole-

cell antitubercular activity yet reported for this new class of

antibacterial agents (MIC₉₉of 49 nM in iron-depleted condi-

tions), with good selectivity. This result compares favorably

against isoniazid, a ﬁrst-line antitubercular drug that displayed

an MIC₉₉of 0.18 µM under the assay conditions.¹⁴Even more

signiﬁcantly, cyclopropyl analogue 16 displayed enhanced

selectivity with an MIC₉₉of 98 nM under iron-deﬁcient

conditions but 6.25 µM under iron-rich conditions, representing

a 64-fold difference in activity, a phenotype concordant with

the designed mechanism of action. The selectivity factor, S, in

Table 1 is deﬁned as the ratio of MIC₉₉values in iron-replete

and iron-deﬁcient conditions. Thus, cyclopropyl derivative 16

provides a 16-fold enhancement in selectivity relative to Sal-

AMS 6.

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5355

of 6 have in the majority of cases resulted in drastic losses in

binding afﬁnity.^21,41–44The high selectivity observed for several

potent MbtA inhibitors, including compound 16 but also

compounds 10-13 and 24-26, which displayed potent submi-

cromolar MIC₉₉values, makes these compounds very promising

antitubercular agents.

shown that a related bisubstrate inhibitor incorporating an

acylhydroxamoylsulfamate linkage, which is approximately 2

Å longer than the acylsulfamate linkages employed in Sal-AMS,

displayed simple additivity in binding relative to the corre-

sponding fragments AMP and 2,3-dihydroxybenzoic acid toward

the adenylation enzyme EntE involved in biosynthesis of the

enterobactin siderophore in E. coli.⁵²

Our SAR results using a bisubstrate inhibitor are consistent

with the extensive substrate speciﬁcity studies of aminoacyl

adenylation domains embedded in multifunctional nonribosomal

peptide synthetases (NRPSs) reported by von Do¨hren and co-

workers using a series of ATP analogues.⁴⁵Signiﬁcantly, ATP

analogues modiﬁed at the C-2 position of adenine were

competent substrates whereas C-8 modiﬁed analogues were not

accepted by the various NRPSs investigated. These co-workers

also demonstrated that deletion of the N-7 nitrogen atom of

adenine led to a marginal reduction in activity. Consequently,

the SAR observed for MbtA, a stand-alone aryl acid adenylating

enzyme (AAAE), is expected to be applicable to the abundant

aminoacyl adenylation domains found in multifunctional NRPSs.

The tight-binding of the intermediate acyladenylate (Sal-

AMP, 2) is necessary to prevent diffusional loss to the bulk

solvent and adventitious hydrolysis of the mixed carboxylic-

phosphoric acid anhydride, since this intermediate must be

channeled to a downstream protein. Unlike the stand-alone aryl

adenylating enzyme MbtA, most NRPS adenylation domains

are embedded with carrier domains in multifunctional proteins

so that transfer to downstream carrier domains is entropically

favored.^53–55Further, many adenylating enzymes such as CoA

ligases employ abundant diffusible small molecules. In both

cases, tight binding of the intermediate acyladenylate may not

be necessary because the effective concentration of the nucleo-

philic acceptor residue is expected to be signiﬁcantly higher.

Consequently, simple bisubstrate inhibitors toward such enzymes

are likely to be intrinsically less potent. Indeed the K_i^appvalue

of cysteyl-AMS for the cysteine adenylation domain in the

multifunctional NRPS module known as HMWP2 from Yersinia

pestis is only 0.24 µM.¹⁶

With the exception of compounds 28 and 29, whole-cell

activity of the nucleobase-modiﬁed analogues correlated nicely

with the in vitro enzyme inhibition results. As previously

suggested, the difference between MIC₉₉and K_i^applikely reﬂects

limited permeability across the mycobacterial cell envelope,

which provides intrinsic resistance to many antibiotics.²¹

Furthermore, if MbtA does not catalyze the rate-limiting

biosynthetic step of the mycobactins, then it may be necessary

to fully abrogate MbtA activity to have an appreciable effect

on mycobactin production.²¹The low whole-cell activity of

compounds 28 and 29, despite their high potency against MbtA,

is most likely due to their inability to use a transporter for uptake

in M. tuberculosis. We hypothesize that the transporter still

recognizes and imports compounds such as 24-26, with a

signiﬁcant change introduced by the bulky substituents in the

C-2 position of the adenine ring. However, the more signiﬁcant

structural change caused by the introduction of a second phenyl

ring in compounds 27-29 probably prevents recognition by

these putative transporters. M. tuberculosis encodes several ABC

transporters (ATP-depending binding cassette) that have been

unambiguously assigned as nucleoside importers.⁴⁶Other

candidate transporters include porins such as MshA and the

mycobactin transporters IrtAB.^47,48

Mechanism of Action. Siderophore biosynthesis is not

required for growth under iron-replete conditions; thus, antitu-

bercular activity under these conditions suggests off-target

binding and subsequent inhibition of alternate biochemical

pathways.¹²Despite the off-target binding of the nucleoside

derivatives described herein, these compounds remain very

promising antitubercular agents. Antitubercular agents that

disrupt multiple biosynthetic pathways may actually be preferred

over single-target therapeutics. Coincidently, many of the

currently used antitubercular agents including isoniazid,⁵⁶

ethionamide, p-aminosalicylic acid, and cycloserine have been

implicated to possess multiple mechanisms of action.⁵⁷M.

tuberculosis encodes for more than 60 adenylation enzymes that

represent potential off-target candidates proteins including 20

aminoacyl tRNA synthestases involved in protein synthesis,⁵⁸

34 FadDs involved in lipid metabolism,³⁶MshC that catalyzes

the third step in mycothiol biosynthesis,⁵⁹PanC that catalyzes

the second step in biosynthesis of coenzyme A (CoASH),⁶⁰

LigA, a NAD-dependent DNA ligase,⁶¹and 5 adenylation

domains embedded in NRPS modules from the mycobactin-

1¹¹and nrp loci.⁶²Several of the most logical potential

candidates were evaluated including the aminoacyl tRNA

synthetases involved in protein synthesis as well as a panel of

fatty acid adenylating enzymes required for lipid metabolism.

Signiﬁcantly, Sal-AMS did not inhibit the functionally related

aminoacyl tRNA synthetases using an in vitro luciferase

translation assay. Additionally, we evaluated the parent com-

pound Sal-AMS 6 against a panel of fatty acid adenylating

(FadD) enzymes, which carry out the analogous adenylation of

fatty acids in M. tuberculosis.³⁶The FadDs can be grouped into

two classes: acyl-CoA synthetases (ACSs) involved in fatty acid

catabolism; long chain fatty acyl-AMP ligases involved in fatty

acid biosynthesis. The long chain acyl-AMP ligases FadD26

and FadD28 were investigated because these are involved in

production of the dimycocerosyl phthiocerols (PDIM), which

are important virulence factors.^36,63–65Similarly, the acyl-CoA

synthetases FadD17 and FadD19 were investigated, since these

have been implicated in mycobacterial persistence via catabolism

of cholesterol.⁶⁶The in vitro ﬁndings that 6 was inactive against

Bisubstrate Inhibition. Sal-AMS and the derivatives de-

scribed herein are considered bisubstrate inhibitors, since these

simultaneously occupy both salicylic acid and nucleoside

binding sites.⁴⁹Bisubstrate inhibitors (A-B) can realize sub-

stantial enhancement in binding energy compared to the sum

of the Gibbs binding energies of the respective fragments (A

and B) as ﬁrst articulated by Jencks because of a smaller entropy

barrier to binding of A-B compared to A + B.⁵⁰In most cases,

bisubstrate inhibitors fail to even display simple additivity in

binding, as linking each fragment typically results in loss of

some interaction energy with the corresponding enzyme. In the

present case, the acylsulfamate closely mimics the native

acylphosphate linkage maintaining all of the key H-bond and

electrostatic interactions with the active site. Isothermal calo-

rimetry revealed the true potency of these inhibitors showing

that 2-phenyl 26 binds with an astonishing K_Dof 0.27 pM or

∆G⁰) -17.5 kcal/mol wherein binding is primarily enthalpy

driven with a minor entropic contribution. The Gibbs binding

energy of 26 is 4 kcal/mol more than the sum of the free energy

of binding of the respective acyladenylate fragments salicylic

acid and AMP (∆G⁰_AMP) -5.0 kcal/mol, ∆G⁰_Sal) -7.9 kcal/

mol).⁵¹By contrast, Callahan, Wolfenden and co-workers have

5356 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

all four of the FadDs evaluated clearly demonstrate a promising

level of selectivity. Future efforts will explore systematic

proteome proﬁling of adenylating enzymes to help identify

potential off-target protein targets and measure selectivity.

Other Pathogens That Require Aryl-Capped Sidero-

phores. Although we have focused on M. tuberculosis, many

other pathogens utilize related aryl-capped siderophores for iron

acquisition and we expect AAAE inhibitors to be effective

against a broad array of clinically relevant pathogens.⁶⁷For

example, Yersinia pestis (plague) and Klebsiella pneumoniae

(opportunistic infections) synthesize yersiniabactin,^68,69Bacillus

anthracis (anthrax) makes petrobactin,^70,71enteric bacteria such

as Escherischia coli and Salmonella enterica serovar Typhimu-

rium produce enterobactin,^72,73Vibrio cholerae (cholera) syn-

thesizes vibriobactin,⁷⁴Vibrio Vulniﬁcus (septic infections)

synthesizes vulnibactin,⁷⁵Pseudomonas aeruginosa (opportu-

nistic infections) makes pyochelin,⁷⁶and Acinetobacter bau-

mannii (opportunistic infections) produces acinetobactin.⁷⁷The

requirement for siderophore synthesis has been demonstrated

to be essential for virulence in in vivo models for invasive

pathogens such as Y. pestis, K. pneumoniae, V. Vulniﬁcus, B.

anthracis, and S. typhimurium.^68–70,75Surface pathogens such

as V. cholera and P. aeruginosa employ redundant mechanisms

of iron acquisition so that the aryl-capped siderophores are not

required for virulence.^78,79Adenylation inhibitors based on the

prototypical Sal-AMS template have been shown to inhibit the

adenylating enzymes AsbC,⁷¹EntE,⁵²DhbE,¹³and YbtE¹²from

B. anthracis, E. coli, B. subtilis, and Y. pestis. Additionally, in

vitro data against Y. pestis and Y. pseudotuberculosis indicated

that Sal-AMS and derivatives possesses activity against these

Gram-negative organisms.^12,21

siderophore synthesis is not possible. However, the bisubstrate

nucleoside inhibitors described herein hold promise to provide

agents with a narrow therapeutic window against pathogens such

as M. tuberculosis that require aryl-capped siderophores for

virulence, which may in fact be advantageous since it is expected

that beneﬁcial commensal bacteria will not be disturbed.

Experimental Section

Molecular Modeling. A homology model of MbtA was con-

structed on the basis of the crystal structure of the 2,3-dihydroxy-

benzoic acid (DHB) adenylating enzyme DhbE (PDB code

1MDB¹⁵) in complex with the adenylated reaction product of 2,3-

dihydroxy acid (2,3-DHB-AMP), a close analogue of 2, which

serves as the rational basis for 5′-O-[N-(salicyl)sulfamoyl]adenosine

6. A primary sequence alignment was generated for DhbE and

MbtA with ClustalW,⁸¹examined, and edited by hand. Modeller

9v1⁸²was used to generate 10 conformations in the presence of 2

obtained by deleting the 3-hydroxyl from the crystal structure. After

examination, a structure with one of the lowest DOPE and highest

objective scores (Modeller structure rankings) was chosen because

of the reasonable positioning of active site side chains. Hydrogens

were added and minimized with Macromodel 9.1⁸³and the

MMFFs⁸⁴force ﬁeld in a GB/SA⁸⁵implicit solvation model. A

130 000 step conformational search including 18 key active site

side chains and the ligand was undertaken to explore the transla-

tional and torsional energy surface of residues near the binding

pocket. In recognition of the 76% identical active sites, select

hydrogen bonding interactions were restrained through the addition

of harmonic constraints with a large penalty for distances outside

a 2.7-3.5 Å range. A structure that differed from the global

minimum by 1.5 kcal/mol and a torsion of the Asp436 (DhbE

Asp413) side chain was chosen for future modeling studies because

it positions the aspartic acid side chain such that it doubly hydrogen-

bonds the ribosyl moiety (one side chain oxygen hydrogen-bonded

to the 2′-hydroxyl, the other to the 3′-hydroxyl) in agreement with

the DhbE crystal structure.

Conclusion

Molecular modeling studies were used to guide modiﬁcations

to the nucleobase of the parent compound Sal-AMS 6 and clear

trends in the SAR data emerged demonstrating the remarkable

ability to tolerate bulky substituents at the C-2 position and small

alkyl substituents appended at N-6 of the purine but not

modiﬁcation at the C-8 position. The comprehensive series of

analogues conﬁrmed that N-1 and N-3 were dispensable for

activity, while deletion of N-7 results in modest decreases in

potency and biological activity. N-6 cyclopropyl 16 was found

to exhibit the highest selectivity against M. tuberculosis under

iron-rich conditions. Isothermal calorimetry revealed the intrinsic

potency of this class of inhibitors with 2-phenyl 26 displaying

femtomolar enzyme inhibition and the most potent antitubercular

activity yet observed for this class of antitubercular agents.

Attempts to delineate the putative secondary mechanism of

action of this class of compounds under iron-rich conditions

revealed that Sal-AMS does not inhibit the functionally related

aminoacyl t-RNA synthetases or several mycobacterial fatty acid

adenylating enzymes (FadDs) involved in lipid metabolism.

Further, these compounds displayed no overt cytotoxicity

providing large therapeutic indexes. Recent studies have shown

that Sal-AMS 6 is stable in human plasma and human liver

microsomes.⁸⁰Taken together, these results provide a promising

proﬁle for the described 5′-O-[N-(salicyl)sulfamoyl]nucleoside

inhibitors and the modiﬁcations described herein have enabled

signiﬁcant improvements in potency, selectivity, and lipophi-

licity of this new class of antitubercular agents. Nucleoside

inhibitors based on Sal-AMS have great potential as novel

antibacterial agents targeting siderophore biosynthesis. More

than 500 structurally different siderophores have been isolated.

Thus, the development of a broad-spectrum agent targeting

Molecular Dynamics. The MbtA homology model, with the

ligand absent, was prepared for molecular dynamics simulation

using VMD.⁸⁶Charges were neutralized through addition of Na⁺

and Cl^-ions, and the model was soaked in a water box extending

at least 15 Å beyond any atom of the structure in each direction.

The entire model was ﬁrst minimized in NAMD,⁸⁷using the

CHARMM^88,89force ﬁeld, and then warmed from a temperature

of 5 to 300 K over 60 ps. Following the model preparation a 32 ns

simulation was completed.

QM/MM Studies. QM/MM geometry optimizations were per-

formed in Qsite⁹⁰using the homology model described above.

Ligands were treated at the B3LYP^91–93/6-31G(d)^94,95level of

theory polarized by OPLS 2005⁹⁶charges in the positions of the

protein atoms. The lack of covalent bonds between the two regions

circumvented the problem of treating link atoms. Both ligand and

protein were allowed to minimize freely.

Chemistry General Procedures. All commercial reagents

(Sigma-Aldrich, Acros) were used as provided unless otherwise

indicated. Tubercidin 30 was kindly provided by Prof. Herbert

Nagasawa (VA Medical Center, Minneapolis, MN). 8-Bromoad-

enosine was obtained from Berry and Associates (Dexter, MI).

Sulfamoyl chloride was prepared by the method of Heacock except

that this was used directly without recrystallization.⁹⁷N-Hydrox-

ysuccinimidyl 2-(methoxymethoxy)benzoate 33 was prepared as

described.²¹An anhydrous solvent dispensing system (J. C. Meyer)

using two packed columns of neutral alumina was used for drying

THF, DMF, and CH₂Cl₂, and the solvents were dispensed under

argon. Anhydrous DME was purchased from Aldrich and used as

provided. Flash chromatography was performed with silica P grade

silica gel 60 (Silicycle) or with an ISCO Combiﬂash Companion

puriﬁcation system with the prepacked silica gel cartridges with

the indicated solvent system. All reactions were performed under

an inert atmosphere of dry Ar or N₂in oven-dried (150 °C)

1

13

glassware. H and C NMR spectra were recorded on a Varian

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5357

600 MHz spectrometer. Proton chemical shifts are reported in ppm

from an internal standard of residual chloroform (7.26 ppm) or

methanol (3.31 ppm), and carbon chemical shifts are reported using

an internal standard of residual chloroform (77.0 ppm) or methanol

(49.1 ppm). Proton chemical data are reported as follows: chemical

shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q ) quartet,

p ) pentet, m ) multiplet, br ) broad), coupling constant,

integration. High resolution mass spectra were obtained on Agilent

TOF II TOF/MS instrument equipped with either an ESI or APCI

interface. Optical rotations were measured on a Rudolph Autopol

III polarimeter. Melting points were measured on an electrothermal

Mel-Temp manual melting point apparatus and are uncorrected.

Preparative HPLC was performed on a Varian Microsorb MV 100-8

C18 column (41.4 mm × 250 mm, 8 µm particle size) operating at

40 mL/min with detection at 254 nm and the following mobile

phases (solvent A, 50 mM Et₃NH·HCO₃; solvent B1, 9:1 MeCN/

H₂O; solvent B2, MeOH) and the indicated HPLC conditions

(methods 1 and 2). Method 1 involves elution with solvent A and

20-100% linear gradient of solvent B1 over 30 min. Method 2

involves elution with solvent A and 20-100% linear gradient of

solvent B2 over 30 min. HPLC purity of synthesized compounds

was determined using a Varian Microsorb MV 100-8 C18 column

(4.6 mm × 250 mm, 8 µm particle size) operating at 0.5 mL/min

with detection at 254 nm and the conditions described in the

Supporting Information.

concentrated under reduced pressure. Puriﬁcation by ﬂash chro-

matography (EtOAc/MeOH/Et₃N) afforded the title compound.

General Procedure for Ion Exchange. A solution of a nucleoside

triethylammonium salt (0.25 mmol, 1 equiv) in H₂O (0.5 mL) was

added to a short column (10 mm × 50 mm, Dowex 50WX8-100-

Na⁺) and incubated for 10 min before eluting with H₂O (20 mL).

The fractions containing the product were lyophilized to afford the

sodium salt as a ﬂocculent white solid. The Dowex cation exchange

resin was converted to the sodium form by sequentially washing

the column with MeOH (50 mL), H₂O (50 mL), 1 N aqueous NaOH

(25 mL), and H₂O (100 mL).

7-Deaza-2′,3′-O-isopropylideneadenosine (31). 7-Deazaadenosine

(tubercidin) 30 (300 mg, 1.12 mmol, 1.00 equiv) was converted to

the title compound using the general procedure for acetonide

protection. Puriﬁcation by ﬂash chromatography (99:1 EtOAc/

MeOH) afforded the title compound (304 mg, 88%) as a colorless

oil: R_f) 0.6 (3:17 MeOH/EtOAc); [R]²_D⁰-220 (c 0.230, MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.33 (s, 3 H), 1.58 (s, 3H), 3.68

(dd, J ) 12.0, 4.2 Hz, 1H), 3.76 (dd, J ) 12.0, 3.0 Hz, 1H), 4.25

(app q, J ) 3.6 Hz, 1H), 4.96 (dd, J ) 6.0, 2.4 Hz, 1H), 5.12 (dd,

J ) 6.0, 4.2 Hz, 1H), 6.12 (d, J ) 4.2 Hz, 1H), 6.57 (d, J ) 3.6

Hz, 1H), 7.26 (d, J ) 3.6 Hz, 1H), 8.06 (s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 24.4, 26.5, 62.4, 81.4, 83.9, 85.6, 91.5, 99.8, 104.0,

114.1, 123.3, 149.3, 151.0, 157.9; MS (APCI+) calcd for

C₁₄H₁₉N₄O₄[M + H]⁺307.1, found 307.1.

General Procedure for Acetonide Protection. A mixture of

nucleoside (1.0 equiv), 2,2-dimethoxypropane (18 equiv), and

camphorsulfonic acid·H₂O (1.0 equiv) in acetone (0.1 M) was

stirred at room temperature for 16 h. Solid NaHCO₃(3.0 equiv)

was added, and the heterogeneous mixture was stirred for 30 min.

The mixture was then partitioned between CHCl₃(25 mL/mmol)

and H₂O (10 mL/mmol). The aqueous layer was back-extracted

with CHCl₃(3 × 25 mL), and the combined organic extracts were

dried (Na₂SO₄) and concentrated. Puriﬁcation by ﬂash chromatog-

raphy (EtOAc/hexane) afforded the title compound.

7-Deaza-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (32).

Compound 31 (360 mg, 1.18 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (200:1 EtOAc/MeOH) af-

forded the title compound (350 mg, 79%): R_f) 0.7 (85:15 EtOAc/

MeOH); [R]²_D⁰-8.0 (c 1.2, MeOH); ¹H NMR (600 MHz, CD₃OD)

δ 1.40 (s, 3H), 1.63 (s, 3H), 4.28 (dd, J ) 12.0, 4.8 Hz, 1H), 4.32

(dd, J ) 10.2, 4.6 Hz, 1H), 4.46 (app q, J ) 3.6 Hz, 1H), 5.08 (dd,

J ) 6.0, 2.4 Hz, 1H), 5.23 (dd, J ) 6.0, 3.0 Hz, 1H), 6.32 (d, J )

3.0 Hz, 1H), 6.66 (d, J ) 3.6 Hz, 1H), 7.32 (d, J ) 3.6 Hz, 1H),

8.13 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 25.7, 27.6, 70.1,

82.7, 84.3, 85.6, 91.2, 101.7, 104.9, 115.8, 123.8, 151.2, 152.7,

159.1; MS (APCI+) calcd for C₁₄H₂₀N₅O₆S [M + H]⁺386.1, found

386.1

7-Deaza-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine Tri-

ethylammonium Salt (7). Compound 32 (340 mg, 0.88 mmol, 1.0

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N) afforded the

title compound (168 mg, 41%) as a white solid: R_f) 0.3 (4:1

EtOAc/MeOH); [R]²_D⁰-7.71 (c 1.27, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.26 (t, J ) 7.3 Hz, 9H), 3.15 (q, J ) 7.3 Hz, 6H),

4.25-4.27 (m, 1H), 4.34-4.38 (m, 3H), 4.52 (t, J ) 5.7 Hz, 1H),

6.26 (d, J ) 6.3 Hz, 1H), 6.61 (d, J ) 3.7 Hz, 1H), 6.78-6.81 (m,

2H), 7.30 (t, J ) 7.7 Hz, 1H), 7.54 (d, J ) 3.7 Hz, 1H), 7.95 (d,

J ) 7.7 Hz, 1H), 8.07 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ

9.3, 48.0, 69.9, 72.6, 76.1, 84.0, 88.5, 101.4, 104.8, 118.0, 119.4,

120.8, 123.5, 131.5, 134.5, 151.6, 152.2, 158.9, 162.2, 175.1; HRMS

(ESI-) calcd for C₁₈H₁₈N₅O₈S [M - H]^-464.0882, found

464.0873 (error 1.9 ppm).

General One-Pot Procedure for N⁶-Amination and Isopropy-

lidene Protection. To a suspension of 45 (1.0 equiv) in ethanol (10

mL/mmol) in a glass pressure vessel were added Et₃N (3.0 equiv)

and the amine (1.5 equiv). The glass tube was sealed with the Teﬂon

screw cap and heated at 75 °C for 5-6 h. The pressure vessel was

cooled to room temperature, and the contents were transferred to a

round-bottom ﬂask and concentrated and dried thoroughly under

high vacuum to remove any residual amine. The resulting solid

was suspended in acetone (5 mL/mmol). Dimethoxypropane (2.0

equiv) and p-toluenesulfonic acid (1.0 equiv) were added, and the

mixture was stirred 16 h at room temperature. After completion

solid NaHCO₃(3.0 equiv) was added and the heterogeneous mixture

stirred for 30 min. The mixture was then partitioned between CHCl₃

(75 mL/mmol) and H₂O (15 mL/mmol). The aqueous layer was

back-extracted with CHCl₃(3 × 25 mL), and the combined organic

extracts were dried (Na₂SO₄) and concentrated. Puriﬁcation by ﬂash

chromatography (EtOAc/hexane) afforded the title compound.

General Procedure for Sulfamoylation. To a solution of 2′,3′-

O-isopropylidene protected nucleoside (1 equiv) in DME/THF (1:

1, 50 mL/mmol) at 0 °C was added NaH (1.5 equiv). After 30

min, solid sulfamoyl chloride (1.5 equiv) was added and the mixture

stirred for 16 h at room temperature. The reaction was quenched

at 0 °C with MeOH (30 mL/mmol) and then concentrated under

reduced pressure. Puriﬁcation by ﬂash chromatography afforded

the title compound.

General Procedure for Salicylation. To a solution of 2′,3′-O-

isopropylidene-5′-O-sulfamoyl-nucleoside (1 equiv) in DMF (0.1

M) at 0 °C were added 33 (1.5 equiv) and Cs₂CO₃(3.0 equiv), and

the mixture was stirred for 16 h at room temperature. The mixture

was ﬁltered and then concentrated by rotary evaporation (P ≈ 5

mbar, water bath ∼35 °C). Puriﬁcation by ﬂash chromatography

(10-20% MeOH/EtOAc + 1% Et₃N) afforded the title compound.

General Procedure for TFA Deprotection. To a solution of 5′-

O-{N-[2-methoxymethoxy)benzoyl]sulfamoyl}-2′,3′-O-isopropylidene-

nucleoside (0.2 mmol) was added 80% aqueous TFA (2.5 mL).

The resulting solution was stirred for 30 min at 0 °C and then

(1R)-2- Aza-2- (benzenesulfonyl) bicyclo [2.2.1] hept- 5-en- 3-

one (35).²²To a solution of (1R)-(-)-azabicyclo[2.2.1]hept-5-ene-

3-one (34) (3.00 g, 27.5 mmol) in THF (250 mL) at 0 °C was added

NaH (60% dispersion in mineral oil, 1.32 g, 33.0 mmol, 1.2 equiv)

and the slurry stirred for 30 min at room temperature. Next, neat

benzenesulfonyl chloride (4.21 mL, 33.0 mmol, 1.2 equiv) was

added and the mixture stirred an additional 1.5 h at room

temperature. The resulting dark-black reaction was quenched with

ice (25 g) and then concentrated. The residue was taken up in

CH₂Cl₂(200 mL) and washed with saturated aqueous NaHCO₃(150

mL), dried (Na₂SO₄), and concentrated. Puriﬁcation by ﬂash

chromatography (CH₂Cl₂) afforded the title compound (4.27 g, 62%)

as a white solid: mp 89-91 °C; R_f) 0.35 (1:9 CH₂Cl₂); [R]_D²⁰

1

+10.9 (c 0.110, MeOH); H NMR (600 MHz, CDCl₃) δ 2.18 (d,

J ) 8.4 Hz, 1H), 2.42 (d, J ) 8.4 Hz, 1H), 3.38 (s, 1H), 5.05 (s,

1H), 6.37 (s, 1H), 6.62 (d, J ) 4.8 Hz, 1H), 7.50 (t, J ) 7.8 Hz,

5358 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

2H), 7.61 (t, J ) 7.8 Hz, 1H), 7.91 (d, J ) 7.8 Hz, 2H); ¹³C NMR

(150 MHz, CDCl₃) δ 54.4, 55.5, 65.4, 127.7, 128.9, 133.8, 136.4,

137.7, 139.3, 174.6; HRMS (ESI-) calcd for C₂₆H₂₈N₃O₁₀S [M -

H]^-574.1501, found 564.1498 (error 0.5 ppm).

(150 MHz, CDCl₃) δ 27.2, 35.4, 38.8, 44.5, 61.9, 66.4, 102.3,

116.3, 117.5, 120.3, 123.1, 129.4, 131.1, 136.8, 138.9, 140.4,

178.4.

(1S,2R,3S,5S)-1-{1,2-Dihydroxy-1,2-O-isopropylidene-5-[(piv-

aloyloxy)methyl]cyclopent-3-yl}-4-nitroindole (40) and (1R,2S,3S,5S)-

1-{1,2-Dihydroxy-1,2-O-isopropylidene-5-[(pivaloyloxy)methyl]cy-

clopent-3-yl}-4-nitroindole (39). To a solution of 38 (1.24 g, 3.97

mmol, 1.0 equiv) in THF (40 mL) OsO₄(2.50 mL, 2.5 wt %

solution in t-BuOH, 0.19 mmol, 0.05 equiv) and NMO (931 mg,

7.94 mmol, 2.0 equiv) were added, and the mixture was stirred

2.5 h at room temperature. The reaction was quenched with 10%

aqueous Na₂S₂O₅(20 mL) and then partitioned between EtOAc

(250 mL) and H₂O (100 mL). The aqueous phase was back-

extracted with EtOAc (2 × 125 mL), and the combined organic

extracts were dried (Na₂SO₄) and concentrated to an orange foam.

The crude diol was dissolved in 2,2-dimethoxypropane (50 mL).

Then p-TsOH·H₂O (75 mg, 3.97 mmol, 0.10 mmol) was added

and the resulting dark-brown solution was stirred at room temper-

ature for 24 h. The reaction was quenched with solid NaHCO₃(1.0

g). The mixture was ﬁltered through Celite washing with acetone

(25 mL), and the ﬁltrate was concentrated. Puriﬁcation by ﬂash

chromatography (10-20% EtOAc/hexane) afforded two diastere-

omers 39 (400 mg, 26%) and 40 (371 mg, 24%). Assignment was

(1R,4S)-1-(Benzenesulfonylamino)-4-(hydroxymethyl)cyclopent-

2-ene (36).²²To a solution of lactam 35 (3.99 g, 16.0 mmol, 1.0

equiv) in MeOH (75 mL) at 0 °C was added NaBH₄(1.81 g,

48.0 mmol, 3.0 equiv) in ﬁve portions over 5 min. The mixture

was stirred for 1.5 h at 0 °C and then quenched by the dropwise

addition of saturated aqueous NH₄Cl (40 mL) and partitioned

between EtOAc (300 mL) and H₂O (100 mL). The aqueous layer

was separated and back-extracted with EtOAc (2 × 100 mL),

and the combined organic extracts were dried (Na₂SO₄) and

concentrated. Puriﬁcation by ﬂash chromatography (Et₂O) af-

forded the title compound (4.05 g, 100%) as a viscous colorless

1

oil: R_f) 0.27 (Et₂O); [R]²_D⁰- 36.9 (c 1.94, MeOH); H NMR

(600 MHz, CDCl₃) δ 1.18 (t, J ) 7.2 Hz, 1H), 1.30 (d, J )

14.4 Hz, 1H), 2.17 (dt, J ) 14.4, 9.0 Hz, 1H), 2.66-2.76 (m,

1H), 3.44-3.50 (m, 2H), 3.58 (dd, J ) 10.8, 3.0 Hz, 1H), 4.32

(d, J ) 8.4 Hz, 1H), 5.55 (d, J ) 5.4 Hz, 1H), 5.70 (d, J ) 5.4

Hz, 1H), 7.49 (t, J ) 7.8 Hz, 2H), 7.55 (t, J ) 7.8 Hz, 1H),

7.87 (d, J ) 7.8 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 34.3,

46.1, 58.6, 63.5, 126.9, 129.1, 132.4, 132.5, 135.1, 141.2; HRMS

(ESI-) calcd for C₁₂H₁₅NNaO₃S [M + Na]⁺276.0665, found

276.0677 (error 4.7 ppm).

1

based on H-¹H COSY and NOESY spectra.

Data for 39: R_f) 0.50 (70:30 hexane/EtOAc); [R]²_D⁰+ 49.4 (c

1

0.77, MeOH); H NMR (600 MHz, CDCl₃) δ 1.25 (s, 9H, t-Bu),

(1R,4S)-1-(N-Benzenesulfonyl-N-pivaloylamino)-4-[(pivaloyloxy)m-

ethyl]cyclopent-2-ene (37).²²To a slurry of NaH (6.40 g, 160

mmol, 10.0 equiv) in THF (100 mL) at 0 °C was cannulated a

solution of alcohol 36 (4.05 g, 16.0 mmol, 1.0 equiv) in THF

(100 mL) over 5 min, and the mixture was stirred at room

temperature for 30 min. Next, neat pivolyl chloride (19.7 mL,

160 mmol, 10.0 equiv) was added and the mixture was reﬂuxed

4 h. After the mixture was cooled to room temperature, the

reaction was quenched by the slow addition of saturated aqueous

NH₄Cl (100 mL), extracted with Et₂O (2 × 250 mL), dried

(Na₂SO₄), and concentrated to a yellow oil. Puriﬁcation by ﬂash

chromatography (90:10 hexane/EtOAc) afforded the title com-

pound (5.74 g, 88%) as a white crystalline solid: mp 66-67

°C; R_f) 0.24 (90:10 hexane/EtOAc); [R]²_D⁰-18.1 (c 0.93,

MeOH); ¹H NMR (600 MHz, CDCl₃) δ 1.17 (s, 9H), 1.32 (s,

9H), 1.79 (dt, J ) 13.8, 7.2 Hz, 1H), 2.20 (dt, J ) 13.8, 8.4 Hz,

1H), 2.80-2.90 (m, 1H), 3.94-4.04 (m, 2H), 4.82 (t, J ) 7.8

Hz, 1H), 5.63 (d, J ) 5.4 Hz), 5.79 (d, J ) 5.4 Hz, 1H), 7.51

(t, J ) 7.8 Hz, 2H), 7.60 (t, J ) 7.8 Hz, 1H), 7.89 (d, J ) 7.8

Hz, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 27.1, 28.2, 32.6, 38.7,

43.8, 43.9, 65.2, 66.8, 128.5, 128.8, 131.1, 133.2, 134.2, 139.8,

178.3, 185.9.

1.30 (s, 3H, acetonide), 1.63 (s, 3H, acetonide), 2.21 (q, J ) 12.6

Hz, 1H, H-4′ꢁ), 2.53 (dt, J ) 12.6, 6.0 Hz, 1H, H-4′R), 2.62 (dp,

J ) 12.6, 6.0 Hz, 1H, H-5′), 4.24 (dd, J ) 10.8, 6.0 Hz, 1H, PivO-

CH₂-), 4.30 (dd, J ) 10.8, 6.0 Hz, 1H, PivO-CH₂-), 4.55 (t, J )

6.6 Hz, 1H, H-1′), 4.61 (dd, J ) 7.2, 5.4 Hz, 1H, H-2′), 4.84 (dt,

J ) 12.0, 6.0 Hz, 1H, H-3′), 7.28 (ovlp d, J ) 3.0 Hz, 1H, H-3),

7.30 (ovlp t, J ) 7.8 Hz, 1H, H-6), 7.40 (d, J ) 3.0 Hz, 1H, H-2),

7.92 (d, J ) 7.8 Hz, 1H, H-7), 8.14 (d, J ) 7.8 Hz, 1H, H-5);

NOE ¹H NMR (600 MHz, CDCl₃) (Irr, H-3′), enhancements at

2.53 (H-4′R), 2.62 (H-5′), 7.92 (H-7); ¹³C NMR (150 MHz, CDCl₃)

δ 24.9, 27.2, 27.5, 33.5, 38.9, 43.2, 61.9, 64.4, 81.1, 85.3, 102.7,

114.1, 117.2, 117.8, 120.7, 122.9, 128.2, 138.7, 140.4, 178.3; HRMS

(ESI-) calcd for C₂₂H₂₈N₂NaO₆[M + Na]⁺439.1840, found

439.1841 (error 0.2 ppm).

Data for 40: R_f) 0.38 (70:30 hexane/EtOAc); [R]²_D⁰+ 60.5 (c

1

0.11, MeOH); H NMR (600 MHz, CDCl₃) δ 1.22 (s, 9H, t-Bu),

1.25 (s, 3H, acetonide), 1.50 (s, 3H, acetonide), 2.12-2.15 (m, 1H,

H-4′ꢁ), 2.24-2.35 (m, 2H, H-5′, H-4′R), 4.28 (dd, J ) 10.8, 7.2

Hz, 1H, PivO-CH₂-), 4.38 (dd, J ) 13.8, 6.6 Hz, 1H, PivO-CH₂-),

4.62 (dt, J ) 11.4, 5.4 Hz, 1H, H-3′), 4.73-4.76 (m, 2H, H-1′,

H-2′), 7.25-7.28 (m, 2H, H-3, H-6), 7.59 (d, J ) 3.0 Hz, 1H, H-2),

7.68 (d, J ) 7.8 Hz, 1H, H-7), 8.13 (d, J ) 7.8 Hz, 1H, H-5);

1

NOE H NMR (600 MHz, CDCl₃) 4.62 (Irr, H-3′), enhancement

(1R,4S)-1-{4-[(Pivaloyloxy)methyl]cyclopent-2-ene-1-yl}-4-ni-

troindole (38).²²To a solution of 4-nitroindole (1.31 g, 8.06

mmol, 1.1 equiv) in DMSO (100 mL) was added NaH (60%

dispersion in mineral oil, 322 mg, 8.06 mmol, 1.1 equiv). The

resulting deep-purple solution was stirred for 10 min, and then

Pd₂(dba)₃· CHCl₃(190 mg, 0.183 mmol, 0.025 equiv) and neat

P(O-i-Pr)₃(362 µL, 1.47 mmol, 0.20 equiv) were added

sequentially. Next, a solution of 37 (3.00 g, 7.33 mmol, 1.0

equiv) in THF (100 mL) was cannulated and the mixture stirred

for 3 h at room temperature. The reaction mixture was partitioned

between EtOAc (500 mL) and H₂O (100 mL). The organic phase

was separated and washed successfully with H₂O (2 × 250 mL)

and saturated aqueous NaCl (250 mL), dried (Na₂SO₄), and

concentrated. Puriﬁcation by ﬂash chromatography (6:1 hexane/

EtOAc) afforded the title compound (1.25 g, 55%) as a light-

orange oil: R_f) 0.13 (90:10 hexane/EtOAc); [R]²_D⁰- 20.7 (c

1.60, MeOH); ¹H NMR (600 MHz, CDCl₃) δ 1.18 (s, 9H), 1.60

(td, J ) 13.8, 7.2 Hz, 1H), 2.80 (dt, J ) 13.8, 8.4 Hz, 1H),

3.12-3.22 (m, 1H), 4.04-4.14 (m, 2H), 5.58-5.68 (m, 1H),

5.99-6.02 (m, 1H), 6.15-6.17 (m, 1H), 7.23 (ovlp d, J ) 3.0

Hz, 1H), 7.25 (ovlp t, J ) 7.8 Hz, 1H), 7.39 (d, J ) 3.0 Hz,

1H), 7.76 (d, J ) 7.8 Hz, 1H), 8.12 (d, J ) 7.8, 1H); ¹³C NMR

at 2.12-2.15 (H-4′ꢁ), 2.24-2.35 (H-5′, H-4′R), 4.73-4.76 (H-1′,

H-2′), 7.68 (H-7); ¹³C NMR (150 MHz, CDCl₃) δ 23.7, 25.6, 27.2,

29.9, 38.8, 39.8, 57.6, 63.1, 78.3, 79.1, 101.9, 111.0, 115.5, 117.7,

120.3, 122.8, 131.5, 138.5, 140.5, 178.4; HRMS (ESI-) calcd for

C₂₂H₂₉N₂O₆[M + H]⁺417.2020, found 417.2032 (error 2.9 ppm).

(1R,2S,3S,5S)-1-{1,2-Dihydroxy-5-[(hydroxy)methyl]-1,2-O-iso-

propylidenecyclopent-3-yl}-4-nitroindole (41). A suspension of 39

(264 mg, 0.68 mmol, 1.0 equiv) in MeOH/0.5 M aqueous NaOH

(3:2, 100 mL) was heated at 100 °C for 2 h. The resulting

homogeneous solution was cooled to room temperature and

concentrated to remove MeOH, and the remaining aqueous layer

was partitioned between EtOAc (100 mL) and aqueous 0.5 M

NaH₂PO₄(50 mL). The organic layer was separated, dried

(Na₂SO₄), and concentrated. Puriﬁcation by ﬂash chromatography

(70:30 EtOAc/hexane) afforded the title compound (185 mg, 89%)

as a yellow foam: R_f) 0.24 (70:30 EtOAc/hexane); ¹H NMR (600

MHz, CDCl₃) δ 1.31 (s, 3H), 1.63 (s, 3H), 1.71 (br s, 1H, OH),

2.26-2.36 (m, 1H), 2.46-2.56 (m, 2H), 3.78-3.92 (m, 2H),

4.56-4.68 (m, 2H), 4.80-4.88 (m, 1H), 7.24-7.34 (m, 2H), 7.46

(d, J ) 3.0 Hz, 1H), 7.94 (d, J ) 7.8 Hz, 1H), 8.15 (d, J ) 7.8 Hz,

1H); ¹³C NMR (150 MHz, CDCl₃) δ 24.9, 27.5, 32.8, 45.8, 62.4,

63.3, 81.3, 85.6, 102.6, 113.9, 117.4, 117.8, 120.6, 123.0, 128.5,

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5359

138.8, 140.3; HRMS (ESI-) calcd for C₁₇H₂₀N₂NaO₅[M + Na]⁺

355.1270, found 355.1278 (error 2.3 ppm).

44%) as a light-brown solid: ¹H NMR (600 MHz, CD₃OD) δ 1.29

(t, J ) 7.2 Hz, 9H), 1.84-1.94 (m, 1H), 2.36-2.46 (m, 2H), 3.17

(q, J ) 7.2 Hz, 6H), 4.08 (t, J ) 4.8 Hz, 1H), 4.23-4.31 (m, 3H),

4.75 (q, J ) 9.6 Hz, 1H), 6.37 (dd, J ) 6.0, 1.2 Hz, 1H), 6.52 (d,

J ) 3.6 Hz, 1H), 6.71-6.81 (m, 2H), 6.90-6.94 (m, 2H), 7.29

(td, J ) 7.8, 1.8 Hz, 1H), 7.32 (d, J ) 3.6 Hz, 1H), 7.94 (dd, J )

7.8, 1.8 Hz, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.4, 30.4, 44.0,

48.0, 61.5, 71.5, 73.9, 77.6, 99.6, 102.5, 105.6, 117.9, 119.3, 120.1,

120.9, 123.4, 123.8, 131.5, 134.3, 139.5, 140.8, 162.1, 174.9; HRMS

(ESI-) calcd for C₂₁H₂₂N₃O₇S [M - H]^-460.1184, found

460.1168 (error 3.5 ppm).

(1R,2S,3S,5S)-1-(1,2-Dihydroxy-1,2-O-isopropylidene-5-

{[(sulfamoyl)oxy]methyl}cyclopent-3-yl)-4-nitroindole (42). To a

stirred solution of alcohol 41 (183 mg, 0.61 mmol, 1.0 equiv) in

dimethylacetamide (4.0 mL) at 0 °C was added solid sulfamoyl

chloride (280 mg, 2.42 mmol, 4.0 equiv). The mixture was stirred

for 2.5 h at 0 °C and then partitioned between H₂O (150 mL) and

EtOAc (150 mL). The organic layer was separated and washed with

H₂O (2 × 150 mL), saturated aqueous NaCl (100 mL), dried

(Na₂SO₄), and concentrated. Puriﬁcation by ﬂash chromatography

(40-70% EtOAc/hexane) afforded the title compound (200 mg,

86%) as a yellow oil: ¹H NMR (600 MHz, CDCl₃) δ 1.29 (s, 3H),

1.62 (s, 3H), 2.33 (q, J ) 12.6, 1H), 2.56 (dt, J ) 12.6, 6.6 Hz,

1H), 2.64 (dp, J ) 12.6, 6.0 Hz, 1H), 4.36-4.41 (m, 2H), 4.61 (t,

J ) 6.6 Hz, 1H), 4.65 (t, J ) 6.6 Hz, 1H), 4.82 (dt, J ) 12.6, 6.6

Hz, 1H), 7.24 (d, J ) 3.6 Hz, 1H), 7.29 (t, J ) 7.8 Hz, 1H), 7.45

(d, J ) 3.6 Hz, 1H), 7.92 (d, J ) 7.8 Hz, 1H), 8.12 (d, J ) 7.8 Hz,

1H); ¹³C NMR (150 MHz, CDCl₃) δ 24.9, 27.3, 32.9, 43.1, 61.8,

69.9, 80.2, 85.1, 102.5, 114.4, 117.3, 117.8, 120.6, 122.8, 128.5,

138.6, 140.2; HRMS (ESI+) calcd for C₁₇H₂₁N₃NaO₇S [M + Na]⁺

434.0992, found 434.1014 (error 5.1 ppm).

( 1 R , 2 S , 3 S , 5 S ) - 1 - { 1 , 2 - D i h y d r o x y - 5 - [ ( { N - [ ( 2 -

methoxymethoxy)benzoyl]sulfamoyl}oxy)methyl]-1,2-O-isopropy-

lidenecyclopent-3-yl}-4-nitroindole Triethylammonium Salt (43).

Sulfamate 42 (154 mg, 0.403 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for salicylation.

Puriﬁcation by ﬂash chromatography (9:1 EtOAc/hexanes) provided

the product (180 mg, 77%) as a viscous oil: R_f) 0.40 (7:3 EtOAc/

hexanes); [R]²_D⁰+44.6 (c 0.473, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.28 (ovlp t, J ) 7.2 Hz, 9H), 1.29 (ovlp s, 3H), 1.60

(s, 3H), 2.46 (q, J ) 12.0 Hz, 1H), 2.54 (dt, J ) 12.0, 6.0 Hz, 1H),

2.65 (dp, J ) 12.0, 5.4 Hz, 1H), 3.17 (q, J ) 7.2 Hz, 6H), 3.45 (s,

3H), 4.34-4.40 (m, 2H), 4.69 (t, J ) 6.0 Hz, 1H), 4.75 (t, J ) 6.0

Hz, 1H), 4.95 (dt, J ) 12.6, 6.6 Hz, 1H), 5.16-5.19 (m, 2H), 6.98

(t, J ) 7.8 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 1H), 7.16 (d, J ) 3.0 Hz,

1H), 7.26-7.34 (m, 2H), 7.46 (d, J ) 7.2 Hz, 1H), 7.77 (d, J )

3.0 Hz, 1H), 8.03 (d, J ) 8.4 Hz, 1H), 8.08 (d, J ) 7.8 Hz, 1H);

¹³C NMR (150 MHz, CD₃OD) δ 9.3, 25.5, 27.9, 34.5, 44.5, 47.9,

56.7, 63.6, 70.7, 82.2, 86.8, 96.6, 103.2, 115.1, 117.2, 118.5, 118.9,

121.5, 122.6, 124.1, 130.0, 130.9, 131.3, 132.3, 140.4, 141.4, 155.8,

176.3; HRMS (ESI-) calcd for C₂₆H₂₈N₃O₁₀S [M - H]^-574.1501,

found 574.1498 (error 0.5 ppm).

( 1 R , 2 S , 3 S , 5 S ) - 1 - [ 1 , 2 - D i h y d r o x y - 5 - ( { [ N - ( 2 -

hydroxybenzoyl)sulfamoyl]oxy}methyl)cyclopent-3-yl]-4-nitroin-

dole Triethylammonium Salt (44). To a solution of 43 (33 mg,

0.057 mmol, 1.0 equiv) in MeOH (5 mL) was added p-TSA·H₂O

(12 mg, 0.070 mmol, 1.2 equiv), and the mixture was stirred at 70

°C for 3 h. The mixture was cooled to room temperature, neutralized

with triethylamine (100 µL, 0.71 mmol, 10 equiv), and concentrated.

Puriﬁcation by ﬂash chromatography (1:9 MeOH/EtOAc) afforded

the title compound (13 mg, 46%) as a viscous oil: R_f) 0.29

(EtOAc); [R]²_D⁰+57.1 (c 0.210, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.29 (t, J ) 7.2 Hz, 9H), 1.96-2.02 (m, 1H), 2.42-2.50

(m, 2H), 3.29 (q, J ) 7.2 Hz, 6H), 4.08 (dd, J ) 5.4, 2.4 Hz, 1H),

4.25-4.35 (m, 3H), 4.91-4.96 (m, 1H), 6.77-6.82 (m, 2H), 7.16

(d, J ) 3.0 Hz, 1H), 7.27-7.31 (m, 2H), 7.83 (d, J ) 3.0 Hz, 1H),

7.94 (d, J ) 7.8 Hz, 1H), 7.99 (d, J ) 8.4 Hz, 1H), 8.07 (d, J )

8.4 Hz, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.3, 30.2, 44.2, 48.0,

71.4, 73.9, 77.9, 103.1, 117.9, 118.2, 118.5, 119.4, 120.9, 121.1,

124.0, 131.1, 131.4, 134.4, 140.8, 141.4, 162.2, 174.9 (missing 1

Ar C); HRMS (ESI-) calcd for C₂₁H₂₀N₃O₉S [M - H] 490.0926,

found 490.0922 (error 0.8 ppm).

(1R,2S,3S,5S)-4-Amino-1-{1,2-dihydroxy-5-({[N-(2-

hydroxybenzoyl)sulfamoyl]oxy}methyl)cyclopent-3-yl]indole Tri-

ethylammonium Salt (8). To a solution of compound 44 (5.0 mg,

8.4 µmol, 1.0 equiv) in DMF (2.0 mL) was added 10% w/v Pd/C

(3.0 mg), and the mixture was stirred under H₂(1 atm) at room

temperature for 8 h. The mixture was ﬁltered through Celite and

the ﬁltrate concentrated. Puriﬁcation by ﬂash chromatography (80:

20:1 EtOAc/MeOH/Et₃N) afforded the title compound (2.1 mg,

2′,3′-O-Isopropylideneinosine (46). Inosine 45 (1.00 g, 3.73

mmol, 1.00 equiv) was converted to the title compound using the

general procedure for acetonide protection. Puriﬁcation by ﬂash

chromatography (95:5 EtOAc/MeOH) provided the product (0.87

g, 76%) as a white solid: R_f) 0.5 (1:9 MeOH/EtOAc); [R]²_D⁰-35

1

(c 0.50, DMSO); H NMR (600 MHz, CD₃OD) δ 1.41 (s, 3H),

1.30 (s, 3H), 3.74 (dd, J ) 12.0, 4.8 Hz, 1H), 3.80 (dd, J ) 12.0,

3.6 Hz, 1H), 4.37-4.40 (m, 1H), 5.04 (dd, J ) 6.0, 1.8 Hz, 1H),

5.29 (dd, J ) 6.0, 2.4 Hz, 1H), 6.22 (d, J ) 2.4 Hz, 1H), 8.10 (s,

1H), 8.35 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 25.6, 27.6,

63.4, 83.0, 86.0, 88.6, 92.5, 115.4, 126.0, 141.1, 146.9, 149.6, 158.9;

HRMS (APCI-) calcd for C₁₃H₁₅N₄O₅[M - H]^-307.1048, found

307.1072 (error 7.8 ppm).

2′,3′-O-Isopropylidene-5′-O-(sulfamoyl)inosine (47). Compound

46 (250 mg, 0.81 mmol, 1.0 equiv) was converted to the title

compound using the general procedure for sulfamoylation. Puriﬁca-

tion by ﬂash chromatography (95:5 EtOAc/MeOH) provided the

product (280 mg, 92%) as a white solid: R_f) 0.4 (80:20 EtOAc/

MeOH); [R]²_D⁰-14 (c 0.75, DMSO); ¹H NMR (600 MHz, CD₃OD)

δ 1.35 (s, 3H), 1.56 (s, 3H), 4.23-4.31 (m, 2H), 4.49-4.51 (m,

1H), 5.07-5.09 (m, 1H), 5.36 (d, J ) 3.6 Hz, 1H), 6.21 (s, 1H),

8.07 (s, 1H), 8.20 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.3,

26.2, 68.6, 81.6, 84.5, 84.7, 90.9, 114.4, 124.6, 139.7, 145.9, 148.3,

157.7; HRMS (APCI-) calcd for C₁₃H₁₆N₅O₇S [M - H]^-

386.0776, found 386.0798 (error 5.7 ppm).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]inosine Triethylammo-

nium Salt (9). Compound 47 (40 mg, 0.10 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

salicylation and TFA deprotection. Puriﬁcation by ﬂash chroma-

tography (17:3 EtOAc/MeOH) afforded the title compound (48 mg,

50%) as a white solid: R_f) 0.2 (3:7 MeOH/EtOAc); [R]²_D⁰-11.7

(c 1.40, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.27 (t, J ) 7.2

Hz, 9H), 3.17 (q, J ) 7.2 Hz, 6H), 4.31-4.32 (m, 1H), 4.35-4.38

(m, 1H), 4.41-4.42 (m, 2H), 4.76 (t, J ) 6.0 Hz, 1H), 6.07 (d, J

) 6.0 Hz, 1H), 6.76-6.79 (m, 2H), 7.29 (t, J ) 7.8 Hz, 1H), 7.92

(d, J ) 8.4 Hz, 1H), 8.00 (s, 1H), 8.44 (s, 1H); ¹³C NMR (150

MHz, CDCl₃) δ 9.4, 47.9, 69.7, 72.6, 76.1, 84.8, 89.9, 118.0, 119.4,

120.8, 125.7, 131.4, 134.5, 140.9, 146.9, 150.3, 159.1, 162.1, 174.9;

MS (APCI-) calcd for C₁₇H₁₆N₅O₉S [M - H]^-466.1 found 466.1.

2′,3′-O-Isopropylidene-N⁶,N⁶′-dimethyladenosine (49). To a stirred

solution of 48 (200 mg, 0.61 mmol, 1.00 equiv) in tert-BuOH (20

mL) in a sealed tube was added N,N-dimethylamine (2.0 N in

MeOH, 1.0 mL, 2.0 mmol, 3.3 equiv). The mixture was heated at

85 °C for 3 h, then cooled to room temperature and concentrated.

Puriﬁcation by ﬂash chromatography (1:1 EtOAc/hexane) afforded

the title compound (198 mg, 96%) as white solid: R_f) 0.4 (EtOAc);

1

[R]²_D⁰-62 (c 0.50, MeOH); H NMR (600 MHz, CD₃OD) δ 1.51

(s, 3H), 1.77 (s, 3H), 3.65 (br s, 6H), 3.89 (dd, J ) 12.6, 3.6 Hz,

1H), 4.00 (dd, J ) 12.6, 3.0 Hz, 1H), 4.55-4.57 (m, 1H), 5.18

(dd, J ) 6.6, 1.8 Hz, 1H), 5.34 (dd, J ) 6.6, 4.2 Hz, 1H), 6.21 (d,

J ) 4.2 Hz, 1H), 8.27 (s, 1H), 8.34 (s, 1H); ¹³C NMR (150 MHz,

CDCl₃) δ 25.4, 27.9, 39.1 (br), 63.7, 81.9, 82.8, 86.2, 94.6, 114.0,

121.8, 138.2, 148.9, 151.9, 155.3; MS (APCI+) calcd for

C₁₅H₂₂N₅O₄[M + H]⁺336.2, found 336.1.

2′,3′-O-Isopropylidene-5′-O-sulfamoyl-N⁶,N⁶′-dimethyladenos-

ine (59). Compound 49 (175 mg, 0.52 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (3:2 EtOAc/

hexane) provided the product (190 mg, 88%) as a white solid: R_f

) 0.5 (EtOAc); [R]²_D⁰-27.6 (c 1.30, MeOH); ¹H NMR (600 MHz,

5360 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

CD₃OD) δ 1.42 (s, 3H), 1.65 (s, 3H), 3.51 (br s, 6H), 4.31 (dd, J

) 10.8. 4.8 Hz, 1H), 4.37 (dd, J ) 10.8, 4.2 Hz, 1H), 4.51-4.58

(m, 1H), 5.16 (dd, J ) 6.0, 3.0 Hz, 1H), 5.42 (dd, J ) 6.0. 2.4

1H), 6.26 (d, J ) 2.4 Hz, 1H), 8.19 (s, 1H), 8.24 (s, 1H); ¹³C

NMR (150 MHz, CD₃OD) δ 25.6, 27.5, 39.2, 70.1, 83.0, 85.6, 85.7,

91.7, 115.7, 121.3, 139.5, 151.1, 153.5, 156.2; MS (APCI+) calcd

for C₁₅H₂₃N₆O₆S [M + H]⁺415.1, found 415.1.

0.21 mmol, 1.0 equiv) prepared above was converted to the title

compound using the general procedure for TFA deprotection.

Puriﬁcation by ﬂash chromatography (10:90:1 MeOH/EtOAc/Et₃N)

followed by preparative HPLC using method 2 and conversion to

the sodium salt using the general procedure for ion exchange

afforded the title compound (33 mg, 31%) as a white solid (reported

NMR data are for the TEA salt of the title compound): mp >200

°C (dec); R_f) 0.35 (4:1 EtOAc/MeOH); [R]²_D⁷-22.7 (c 0.233,

MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.25 (t, J ) 7.2 Hz, 9H),

3.09 (br s, 3H), 3.15 (q, J ) 7.2 Hz, 6H), 4.32 (dd, J ) 6.0, 3.0

Hz, 1H), 4.37 (dd, J ) 10.8, 3.6 Hz, 1H), 4.41-4.44 (m, 2H),

4.72 (t, J ) 6.0 Hz, 1H), 6.08 (d, J ) 6.0 Hz, 1H), 6.75-6.79 (m,

2H), 7.28 (td, J ) 7.8, 1.8 Hz, 1H), 7.94 (dd, J ) 7.8, 1.8 Hz, 1H),

8.22 (br s, 1H), 8.44 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.2,

27.7, 47.9, 69.6, 72.4, 76.1, 84.5, 89.2, 117.9, 119.3, 120.6, 120.7,

131.3, 134.4, 140.5, 149.7, 153.9, 156.6, 162.0, 175.0; HRMS

(ESI-) calcd for C₁₈H₁₉N₆O₈S [M - H]^-479.0991, found

479.0996 (1.0 ppm).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-N⁶,N⁶′-dimethyladenos-

ine Triethylammonium Salt (10). Compound 59 (60 mg, 0.143

mmol, 1.0 equiv) was converted to the title compound using the

general procedure for salicylation and TFA deprotection. Puriﬁca-

tion by ﬂash chromatography (90:10:1 EtOAc/MeOH//Et₃N) pro-

vided the product (69 mg, 81%) as a white solid: R_f) 0.4 (3:17

MeOH/EtOAc); [R]²_D⁰-32 (c 0.50, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.24 (t, J ) 7.2 Hz, 9H), 3.05 (q, J ) 7.2 Hz, 6H), 3.47

(br s, 6H), 4.34 (q, J ) 3.6 Hz, 1H), 4.39-4.48 (m, 3H), 4.68 (t,

J ) 5.4 Hz, 1H), 6.08 (d, J ) 5.4 Hz, 1H), 6.76 (ovlp t, J ) 8.4

Hz, H), 6.78 (ovlp d, J ) 8.4 Hz, 1H), 7.29 (t, J ) 7.8 Hz, 1H),

7.95 (d, J ) 8.4, 1H), 8.17 (s, 1H), 8.30 (s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 9.9, 39.3, 47.8, 70.0, 72.3, 76.0, 84.2, 89.6, 118.1,

119.4, 120.3, 121.3, 131.7, 134.8, 139.1, 151.5, 153.2, 156.2, 162.2,

175.5; HRMS (ESI-) calcd for C₁₉H₂₁N₆O₈S [M - H]^-493.1147,

found 493.1146 (error 0.2 ppm).

N⁶-Ethyl-2′,3′-O-isopropylideneadenosine (51). The title com-

pound was synthesized from 48 (400 mg, 1.4 mmol, 1.0 equiv)

and ethylamine (2 N in THF, 1.1 mL, 2.1 mmol, 1.5 equiv) using

the general one-pot procedure for N⁶-amination and isopropylidene

protection. Puriﬁcation by ﬂash chromatography (4:1 EtOAc/

hexanes) provided the product (450 mg, 95%) as a white solid: mp

109-111 °C; R_f) 0.64 (9:1 EtOAc/MeOH); ¹H NMR (600 MHz,

CDCl₃) δ 1.19 (t, J ) 7.2 Hz, 3H), 1.27 (s, 3H), 1.53 (s, 3H), 3.54

(br s, 2H), 3.70 (d, J ) 10.8 Hz, 1H), 3.86 (d, J ) 10.8 Hz, 1H),

4.43 (s, 1H), 5.01 (d, J ) 6.0 Hz, 1H), 5.14 (t, J ) 4.8 Hz, 1H),

5.79 (d, J ) 4.8 Hz, 1H), 7.70 (s, 1H), 8.22 (br s, 1H); ¹³C NMR

(150 MHz, CDCl₃) δ 14.8, 25.2, 27.6, 35.4, 63.2, 81.7, 83.0, 86.1,

93.9, 113.8, 121.0, 139.3, 147.2, 152.7, 155.1; HRMS (ESI+) calcd

for C₁₅H₂₂N₅O₄[M + H]⁺336.1666, found 336.1653 (error 3.9

ppm).

2′,3′-O-Isopropylidene-N⁶-methyladenosine (50). The title com-

pound was synthesized from 48 (500 mg, 1.74 mmol, 1.0 equiv)

and methylamine (2 N in THF, 1.74 mL, 3.48 mmol, 2.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (EtOAc)

provided the product (430 mg, 77%) as a viscous oil: R_f) 0.20

(EtOAc); ¹H NMR (600 MHz, CDCl₃) δ 1.32 (s, 3H), 1.59 (s, 3H),

3.12 (br s, 3H), 3.74 (d, J ) 12.6 Hz, 1H), 3.93 (d, J ) 12.6 Hz,

1H), 4.49 (s, 1H), 5.07 (d, J ) 5.4 Hz, 1H), 5.17 (t, J ) 5.4 Hz,

1H), 5.80 (d, J ) 5.4 Hz, 1H), 7.73 (s, 1H), 8.30 (br s, 1H); ¹³C

NMR (150 MHz, CDCl₃) δ 25.5, 27.6, 27.9, 63.6, 82.0, 83.2, 86.3,

94.5, 114.2, 121.7, 139.6, 147.3, 153.0, 156.1; HRMS (ESI+) calcd

for C₁₄H₂₀N₅O₄[M + H]⁺322.1510, found 322.1520 (error 3.1

ppm).

N⁶-Ethyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (61).

Compound 51 (260 mg, 0.78 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (9:1 EtOAc/hexanes) provided

the product (245 mg, 76%) as a viscous oil: R_f) 0.58 (EtOAc);

¹H NMR (600 MHz, CD₃OD) δ 1.28 (t, J ) 7.2 Hz, 3H), 1.38 (s,

3H), 1.60 (s, 3H), 3.59 (br s, 2H), 4.27 (dd, J ) 10.8, 5.4 Hz, 1H),

4.33 (dd, J ) 10.8, 4.8 Hz, 1H), 4.51 (app dd, J ) 7.8, 4.8 Hz,

1H), 5.12 (dd, J ) 6.0, 2.4 Hz, 1H), 5.40 (dd, J ) 6.0, 2.4 Hz,

1H), 6.22 (d, J ) 2.4 Hz, 1H), 8.20 (s, 1H), 8.24 (br s, 1H); ¹³C

NMR (150 MHz, CD₃OD) δ 15.0, 25.5, 27.4, 36.5, 69.9, 82.9, 85.5,

85.6, 91.7, 115.6, 120.7, 140.8, 149.3, 154.1, 156.0; HRMS (ESI+)

calcd for C₁₅H₂₃N₆O₆S [M + H]⁺415.1394, found 415.1404 (error

2.4 ppm).

2′,3′-O-Isopropylidene-5′-O-sulfamoyl-N⁶-methyladenosine (60).

Compound 50 (420 mg, 1.3 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (4:1 EtOAc/hexane) afforded

the title compound (0.45 mg, 86%) as a viscous oil: R_f) 0.59 (9:1

EtOAc/MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.37 (s, 3H), 1.60

(s, 3H), 3.08 (br s, 3H), 4.27 (dd, J ) 10.8, 5.4 Hz, 1H), 4.33 (dd,

J ) 10.8, 4.2 Hz, 1H), 4.51 (app dd, J ) 7.8, 4.8 Hz, 1H), 5.12

(dd, J ) 6.0, 3.0 Hz, 1H), 5.40 (dd, J ) 6.0, 2.4 Hz, 1H), 6.22 (d,

J ) 2.4 Hz, 1H), 8.18 (s, 1H), 8.25 (br s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 25.5, 27.4, 27.7, 69.9, 82.9, 85.5, 85.6, 91.7, 115.6,

120.9, 140.8, 149.1, 154.1, 156.6; HRMS (ESI-) calcd for

C₁₄H₁₉N₆O₆S [M - H]^-399.1092, found 399.1092 (error 0 ppm).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-N⁶-methyladenosine So-

dium Salt (11). Compound 60 (230 mg, 0.57 mmol, 1.0 equiv) was

coupled to compound 33 using the general procedure for salicy-

lation. Puriﬁcation by ﬂash chromatography (10:90:1 MeOH/

EtOAc/Et₃N) afforded 2′,3′-O-isopropylidene-5′-O-{N-[(2-meth-

oxymethoxy)benzoyl]sulfamoyl}-N⁶-methyladenosinetriethylammonium

salt (320 mg, 84%) as a viscous oil: R_f) 0.50 (4:1 EtOAc/MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.25 (t, J ) 7.2 Hz, 9H), 1.37 (s,

3H), 1.61 (s, 3H), 3.09, (br s, 3H), 3.12 (q, J ) 7.2 Hz, 6H), 3.42

(s, 3H), 4.34 (dd, J ) 10.8, 3.9 Hz, 1H), 4.37 (dd, J ) 10.8, 3.9

Hz, 1H), 4.59-4.60 (m, 1H), 5.16 (s, 2H), 5.22 (dd, J ) 6.0, 2.1

Hz, 1H), 5.41 (dd, J ) 6.0, 3.6 Hz, 1H), 6.25 (d, J ) 3.6 Hz, 1H),

6.97 (t, J ) 7.8 Hz, 1H), 7.12 (d, J ) 7.8 Hz, 1H), 7.27 (t, J ) 7.8

N⁶-Ethyl-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine So-

dium Salt (12). Compound 61 (112 mg, 0.27 mmol, 1.0 equiv) was

coupled to 33 using the general procedure for salicylation and TFA

deprotection. Puriﬁcation by ﬂash chromatography (10:90:1 MeOH/

EtOAc/Et₃N) afforded N⁶-ethyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt as a viscous oil (118 mg, 64%): R_f) 0.53 (9:1 EtOAc/MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.26-1.30 (m, 12H), 1.37 (s, 3H),

1.61 (s, 3H), 3.14 (q, J ) 7.2 Hz, 6H), 3.42 (s, 3H), 3.60 (br s,

2H), 4.33 (dd, J ) 10.8, 3.6 Hz, 1H), 4.37 (dd, J ) 10.8, 3.6 Hz,

1H), 4.59-4.61 (m, 1H), 5.16 (s, 2H), 5.22 (d, J ) 6.0 Hz, 1H),

5.39-5.41 (m, 1H), 6.25 (d, J ) 3.0 Hz, 1H), 6.97 (t, J ) 7.8 Hz,

1H), 7.12 (d, J ) 8.4 Hz, 1H), 7.27 (t, J ) 8.4 Hz, 1H), 7.42 (d,

J ) 7.8 Hz, 1H), 8.24 (br s, 1H), 8.45 (s, 1H); HRMS (ESI-)

calcd for C₂₄H₂₉N₆O₉S [M - H]^-577.1722, found 577.1729 (error

1.2 ppm).

Hz, 1H), 7.42 (dd, J ) 7.8, 1H), 8.25 (br s, 1H), 8.44 (s, 1H); ¹³

C

N⁶-Ethyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-methoxymethoxy)-

benzoyl]sulfamoyl}adenosine triethylammonium salt (70 mg, 0.10

mmol, 1.0 equiv) prepared above was converted to the title

compound using the general procedure for TFA deprotection.

Puriﬁcation by ﬂash chromatography (10:90:1 MeOH/EtOAc/Et₃N)

followed by preparative HPLC using method 2 and conversion to

the sodium salt using the general procedure for ion exchange

afforded the title compound (25 mg, 47%) as a white solid (reported

NMR (150 MHz, CD₃OD) δ 9.2, 25.6, 27.5, 27.6, 47.8, 56.6, 69.9,

83.4, 85.66, 85.75, 91.8, 96.4, 115.2, 117.0, 120.7, 122.5, 129.7,

131.0, 132.4, 140.8, 149.7, 154.0, 155.6, 156.7, 176.8; HRMS

(ESI-) calcd for C₂₃H₂₇N₆O₉S [M - H]^-563.1566, found

563.1572 (error 1.1 ppm).

2′,3′-O-Isopropylidene-5′-O-{N-[(2-methoxymethoxy)benzoyl-

]sulfamoyl}-N⁶-methyladenosine triethylammonium salt (140 mg,

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5361

NMR data are for the TEA salt of the title compound): mp >200

°C (dec); R_f) 0.44 (4:1 EtOAc/MeOH); [R]²_D⁷-30.4 (c 0.184,

MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.26-1.31 (m, 12H), 3.18

(q, J ) 7.2 Hz, 6H), 3.60 (br s, 2H), 4.30-4.36 (m, 1H), 4.37 (dd,

J ) 10.8, 3.0 Hz, 1H), 4.40-4.43 (m, 2H), 4.71 (t, J ) 5.4 Hz,

1H), 6.08 (d, J ) 6.0 Hz, 1H), 6.76-6.80 (m, 2H), 7.29 (td, J )

7.8, 1.2 Hz, 1H), 7.93 (dd, J ) 7.8, 1.2 Hz, 1H), 8.21 (br s, 1H),

8.46 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.2, 15.0, 36.5, 47.9,

69.6, 72.4, 76.1, 84.6, 89.2, 117.9, 119.3, 120.5, 120.6, 131.4, 134.4,

140.5, 150.1, 153.9, 156.0, 162.1, 175.0; HRMS (ESI-) calcd for

C₁₉H₂₁N₆O₈S [M - H]^-493.1147, found 493.1153 (1.2 ppm).

2′,3′-O-Isopropylidene-N⁶-propyladenosine (52). The title com-

pound was synthesized from 48 (420 mg, 1.46 mmol, 1.0 equiv)

and n-propylamine (179 µL, 2.19 mmol, 1.5 equiv) using the general

one-pot procedure for N⁶-amination and isopropylidene protection.

Puriﬁcation by ﬂash chromatography (3:1 EtOAc/hexanes) provided

N⁶-Isopropyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (63). Compound 53(250 mg, 0.72 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (9:1 EtOAc/

hexanes) afforded the title compound (205 mg, 67%) as a viscous

1

oil: R_f) 0.57 (EtOAc); H NMR (600 MHz, CD₃OD) δ 1.30 (d,

J ) 6.6 Hz, 6H), 1.37 (s, 3H), 1.59 (s, 3H), 4.26 (dd, J ) 10.8, 5.4

Hz, 1H), 4.33 (dd, J ) 10.8, 4.8 Hz, 1H), 4.42 (br s, 1H), 4.49-4.52

(m, 1H), 5.12 (dd, J ) 6.6, 3.0 Hz, 1H), 5.40 (dd, J ) 6.6, 2.4 Hz,

1H), 6.22 (d, J ) 2.4 Hz, 1H), 8.20 (s, 1H), 8.25 (br s, 1H); ¹³C

NMR (150 MHz, CD₃OD) δ 22.9, 25.5, 27.4, 43.3, 69.9, 82.9, 85.5,

85.7, 91.7, 115.6, 120.5, 140.7, 149.3, 154.2, 155.4; HRMS (ESI-)

calcd for C₁₆H₂₃N₆O₆S [M - H]^-427.1405, found 427.1416 (error

2.6 ppm).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-N⁶-isopropyladenosine

Sodium Salt (14). Compound 63 (160 mg, 0.37 mmol, 1.0 equiv)

was coupled to 33 using the general procedure for salicylation.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

1

the product (443 mg, 87%): R_f) 0.65 (EtOAc); H NMR (600

MHz, CDCl₃) δ 0.93 (t, J ) 7.2 Hz, 3H), 1.30 (s, 3H), 1.57 (s,

3H), 1.59-1.65 (m, 2H), 3.49 (br s, 2H), 3.71 (d, J ) 11.4, 1H),

3.90 (d, J ) 11.4 Hz, 1H), 4.46 (s, 1H), 5.04 (d, J ) 5.4, 1H), 5.14

(t, J ) 5.4 Hz, 1H), 5.78 (d, J ) 5.4 Hz, 1H), 7.71 (s, 1H), 8.25

(br s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 11.5, 22.9, 25.4, 27.8,

42.5, 63.5, 81.9, 83.2, 86.2, 94.4, 114.1. 121.4, 139.5, 147.3, 152.9,

155.5; MS (APCI-) calcd for C₁₆H₂₂N₅O₄[M - H]^-348.2, found

348.1.

afforded

N⁶-isopropyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (205 mg, 80%) as a viscous oil: R_f) 0.45 (90:10 EtOAc/

MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.24 (t, J ) 7.2 Hz, 9H),

1.30 (d, J ) 6.6 Hz, 6H), 1.37 (s, 3H), 1.61 (s, 3H), 3.13 (q, J )

7.2 Hz, 6H), 3.43 (s, 3H), 4.34 (dd, J ) 11.4, 4.2 Hz, 1H), 4.37

(dd, J ) 11.4, 4.2 Hz, 1H), 4.42 (br s, 1H), 4.59-4.60 (m, 1H),

5.16 (s, 2H), 5.22 (dd, J ) 6.0, 1.8 Hz, 1H), 5.41 (dd, J ) 6.0, 3.0

Hz, 1H), 6.26 (d, J ) 3.0 Hz, 1H), 6.97 (t, J ) 7.8 Hz, 1H), 7.12

(d, J ) 7.8 Hz, 1H), 7.27 (t, J ) 7.8 Hz, 1H), 7.42 (d, J ) 7.8 Hz,

1H), 8.24 (br s, 1H), 8.44 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 9.3, 22.9, 25.6, 27.5, 43.5, 47.7, 56.6, 69.8, 83.4, 85.7, 91.7, 96.4,

115.2, 117.0, 120.2, 122.5, 129.7, 131.0, 132.5, 140.7, 149.7, 154.2,

155.3, 155.6, 176.6 (missing 1 Ar C); HRMS (ESI-) calcd for

C₂₅H₃₁N₆O₉S [M - H]^-591.1879, found 591.1898 (error 3.2 ppm).

N⁶-Isopropyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (203 mg, 0.29 mmol, 1.0 equiv) prepared above was converted

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) followed by preparative HPLC using method 2 and

conversion to the sodium salt using the general procedure for ion

exchange afforded the title compound (83 mg, 54%) as a white

solid (reported NMR data are for the TEA salt of the title

compound): mp >200 °C (dec); R_f) 0.53 (4:1 EtOAc/MeOH);

2′,3′-O-Isopropylidene-5′-O-sulfamoyl-N⁶-propyladenosine (62).

Compound 52 (350 mg, 1.0 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (99:1 EtOAc/hexanes) af-

forded the title compound (321 mg, 75%) as a viscous oil: R_f)

1

0.7 (90:10 EtOAc/MeOH); H NMR (600 MHz, CD₃OD) δ 1.01

(t, J ) 7.2 Hz, 3H), 1.38 (s, 3H), 1.60 (s, 3H), 1.62-1.70 (m, 2H),

3.52 (br s, 2H), 4.25 (dd, J ) 10.8, 4.8 Hz, 1H), 4.33 (dd, J )

10.8, 4.8 Hz, 1H), 4.51 (app dd, J ) 7.8, 4.8 Hz, 1H), 5.13 (dd, J

) 6.0, 2.4 Hz, 1H), 6.23 (d, J ) 2.4 Hz, 1H), 8.21 (s, 1H), 8.24

(br s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 11.8, 23.8, 25.6, 27.5,

43.5, 70.0, 83.0, 85.6, 85.8, 91.8, 115.6, 120.8, 140.8, 149.5, 154.2,

156.3; MS (ESI+) calcd for C₁₆H₂₂N₅O₃[M - SO₂NH₂]⁺332.2,

found 332.2.

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-N⁶-propyladenosine Tri-

ethylammonium Salt (13). Compound 62 (200 mg, 0.47 mmol, 1.00

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (95:5 EtOAc/MeOH) afforded the title

compound (82 mg, 85%) as a viscous oil: R_f) 0.3 (95:5 EtOAc/

MeOH); [R]²_D⁰-31.0 (c 0.101, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.01 (t, J ) 7.2 Hz, 3H), 1.25 (t, J ) 7.2 Hz, 9H),

1.64-1.76 (m, 2H), 3.16 (q, J ) 7.2 Hz, 6H), 3.52 (br s, 2H),

4.30-4.34 (m, 1H), 4.35-4.44 (m, 3H), 4.70 (t, J ) 6.0 Hz, 1H),

6.08 (d, J ) 6.0 Hz, 1H), 6.74-6.82 (m, 2H), 7.28 (t, J ) 7.2 Hz,

1H), 7.93 (d, J ) 7.8, 1H), 8.20 (br s, 1H), 8.45 (s, 1H); ¹³C NMR

(150 MHz, CD₃OD) δ 9.3, 11.8, 23.8, 43.6 (br), 48.0, 69.7, 72.5,

76.2, 84.7, 89.3, 118.0, 119.4, 120.6, 120.7, 131.5, 134.5, 140.5,

154.0, 156.3 (br), 162.1, 175.1 (missing 1 Ar C); HRMS (ESI-)

calcd for C₂₀H₂₃N₆O₈S [M - H]^-507.1304, found 507.1315 (error

2.1 ppm).

1

[R]²_D⁷-23.9 (c 0.180, MeOH); H NMR (600 MHz, CD₃OD) δ

1.25 (t, J ) 7.2 Hz, 9H), 1.29 (d, J ) 6.6 Hz, 6H), 3.15 (q, J ) 7.2

Hz, 6H), 4.32-4.34 (m, 1H), 4.38 (dd, J ) 10.8, 3.6 Hz, 1H),

4.41-4.44 (m, 3H), 4.71 (t, J ) 5.4 Hz, 1H), 6.09 (d, J ) 6.0 Hz,

1H), 6.75-6.79 (m, 2H), 7.28 (t, J ) 7.8 Hz, 1H), 7.94 (d, J ) 8.4

Hz, 1H), 8.21 (br s, 1H), 8.43 (s, 1H); ¹³C NMR (150 MHz,

CD₃OD) δ 9.1, 22.9, 43.6, 47.8, 69.7, 72.3, 75.9, 84.4, 89.2, 117.9,

119.3, 120.3, 120.6, 131.3, 134.4, 140.4, 150.0, 154.0, 155.3, 162.0,

174.7; HRMS (ESI-) calcd for C₂₀H₂₃N₆O₈S [M - H]^-507.1304,

found 507.1307 (error 0.6 ppm).

N⁶-Isobutyl-2′,3′-O-isopropylideneadenosine (54). The title com-

pound was synthesized from 64 (500 mg, 1.74 mmol, 1.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (90:10

EtOAc/hexane) afforded the title compound (540 mg, 86%) as a

N⁶-Isopropyl-2′,3′-O-isopropylideneadenosine (53). The title

compound was synthesized from 48 (400 mg, 1.4 mmol, 1.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (9:1

EtOAc/hexanes) afforded the title compound (420 mg, 86%) as a

1

viscous oil: R_f) 0.40 (EtOAc); H NMR (600 MHz, CDCl₃) δ

0.95 (d, J ) 6.6 Hz, 6H), 1.33 (s, 3H), 1.60 (s, 3H), 1.91 (hept, J

) 6.6 Hz, 1H), 3.43 (br s, 2H), 3.74 (d, J ) 12.6 Hz, 1H), 3.93 (d,

J ) 12.6 Hz, 1H), 4.49 (s, 1H), 5.07 (d, J ) 6.0 Hz, 1H), 5.17 (t,

J ) 6.0 Hz, 1H), 5.80 (d, J ) 6.0 Hz, 1H), 7.73 (s, 1H), 8.28 (br

s, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 20.4, 25.5, 27.9, 28.8, 48.2,

63.7, 82.0, 83.2, 86.3, 94.6, 114.1, 121.5, 139.6, 147.4, 153.0, 155.8;

HRMS (ESI+) calcd for C₁₇H₂₆N₅O₄[M + H]⁺364.1979, found

364.1993 (error 3.8 ppm).

1

white solid: mp 133-135 °C; R_f) 0.44 (EtOAc); H NMR (600

MHz, CDCl₃) δ 1.19 (d, J ) 6.6 Hz, 6H), 1.28 (s, 3H), 1.54 (s,

3H), 3.70 (d, J ) 11.4 Hz, 1H), 3.87 (d, J ) 11.4 Hz, 1H), 4.40

(br s, 1H), 4.43 (s, 1H), 5.02 (d, J ) 6.0 Hz, 1H), 5.14 (t, J ) 5.4

Hz, 1H), 5.78 (d, J ) 5.4 Hz, 1H), 7.71 (s, 1H), 8.22 (br s, 1H);

¹³C NMR (150 MHz, CDCl₃) δ 22.8, 25.2, 27.6, 42.4, 63.3, 81.7,

83.0, 86.1, 94.1, 113.8, 121.1, 139.3, 147.3, 152.8, 154.5; HRMS

(ESI+) calcd for C₁₆H₂₄N₅O₄[M + H]⁺350.1823, found 350.1820

(error 0.9 ppm).

N⁶-Isobutyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (64).

Compound 54 (520 mg, 1.4 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (8:1 EtOAc/hexanes) afforded

5362 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

the title compound (510 mg, 82%) as a viscous oil: R_f) 0.55

2.97 (br s, 1H), 4.30 (dd, J ) 10.8, 5.4 Hz, 1H), 4.36 (dd, J )

10.8, 5.4 Hz, 1H), 4.55 (td, J ) 5.4, 2.4 Hz, 1H), 5.16 (dd, J )

6.0, 2.4 Hz, 1H), 5.45 (dd, J ) 6.6, 2.4 Hz, 1H), 6.27 (d, J ) 2.4

Hz, 1H), 8.24 (s, 1H), 8.33 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 7.7, 24.6 (br), 25.6, 27.5, 70.0, 83.0, 85.6, 85.8, 91.9, 115.7, 121.0,

141.3, 149.9 (br), 154.1, 157.2; HRMS (APCI-) calcd for

C₁₆H₂₁N₆O₆S [M - H]^-425.1249, found 425.1281 (error 7.5).

N⁶-Cyclopropyl-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Triethylammonium Salt (16). Compound 65 (150 mg, 0.352

mmol, 1.00 equiv) was converted to the title compound using the

general procedure for salicylation and TFA deprotection. Puriﬁca-

tion by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N) af-

forded the title compound (84 mg, 39%): R_f) 0.3 (90:10 EtOAc/

MeOH); [R]²_D⁰-46.4 (c 0.501, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 0.65-0.67 (m, 2H), 0.88-0.92 (m, 2H), 1.09 (t, J )

1.2 Hz, 9H), 2.65 (q, J ) 7.2 Hz, 6H), 2.96 (br s, 1H), 4.32-4.35

(m, 1H), 4.39 (dd, J ) 10.8, 3.0 Hz, 1H), 4.25-4.44 (m, 2H), 4.72

(t, J ) 5.4 Hz, 1H), 6.11 (d, J ) 5.4 Hz, 1H), 6.78 (ovlp t, J ) 7.8

Hz, 1H), 6.80 (ovlp d, J ) 8.4 Hz, 1H), 7.29 (t, J ) 7.8 Hz, 1H),

7.95 (d, J ) 7.8 Hz, 1H), 8.28 (br s, 1H), 8.48 (br s, 1H); ¹³C

NMR (150 MHz, CD₃OD) δ 7.7, 10.9, 22.2 (br), 47.7, 69.7, 72.5,

76.2, 84.7, 89.4, 118.0, 119.3, 120.7, 120.8, 131.5, 134.4, 140.9,

153.9, 157.2, 162.2, 175.2 (missing 1 aryl carbon); HRMS (ESI-)

calcd for C₂₀H₂₁N₆O₈S [M - H]^-505.1147, found 505.1161 (error

2.8 ppm).

1

(EtOAc); H NMR (600 MHz, CD₃OD) δ 1.01 (d, J ) 6.6 Hz,

6H), 1.40 (s, 3H), 1.61 (s, 3H), 1.99 (hept, J ) 6.6 Hz, 1H), 3.41

(br s, 2H), 4.26 (dd, J ) 10.8, 4.8 Hz, 1H), 4.33 (dd, J ) 10.8, 4.8

Hz, 1H), 4.51-4.53 (m, 1H), 5.14 (dd, J ) 6.0, 3.0 Hz, 1H), 5.42

(dd, J ) 6.0, 2.4 Hz, 1H), 6.24 (d, J ) 2.4 Hz, 1H), 8.23 (s, 1H),

8.25 (br s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 20.6, 25.5, 27.6,

29.8, 49.7, 70.1, 83.2, 85.7, 85.9, 91.9, 115.8, 120.8, 140.9, 149.6,

154.3, 156.6; HRMS (ESI-) calcd for C₁₇H₂₆ClN₆O₆S [M + Cl]^-

477.1329, found 477.1331 (error 0.4 ppm).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-N⁶-isobutyladenosine So-

dium Salt (15). Compound 64 (160 mg, 0.36 mmol, 1.0 equiv) was

coupled to 33 using the general procedure for salicylation. Puriﬁca-

tion by ﬂash chromatography (90:10:1 EtOAc/MeOH//Et₃N) af-

forded

N⁶-isobutyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (170 mg, 67%) as a viscous oil: R_f) 0.51 (19:1 EtOAc/MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.01 (d, J ) 6.6 Hz, 6H), 1.28 (t,

J ) 7.2 Hz, 9H), 1.38 (s, 3H), 1.62 (s, 3H), 1.98 (hept, J ) 6.6

Hz, 1H), 3.17 (q, J ) 7.2 Hz, 6H), 3.40 (br s, 2H), 3.42 (s, 3H),

4.33 (dd, J ) 10.8, 3.6 Hz, 1H), 4.38 (dd, J ) 10.8, 3.6 Hz, 1H),

4.59-4.61 (m, 1H), 5.16 (s, 2H), 5.23 (dd, J ) 6.0, 1.8 Hz, 1H),

5.41 (dd, J ) 6.0, 3.0 Hz, 1H), 6.26 (d, J ) 3.0 Hz, 1H), 6.97 (t,

J ) 7.8 Hz, 1H), 7.12 (d, J ) 7.8 Hz, 1H), 7.27 (t, J ) 7.8 Hz,

1H), 7.42 (d, J ) 7.8 Hz, 1H), 8.23 (br s, 1H), 8.47 (s, 1H); ¹³C

NMR (150 MHz, CD₃OD) δ 9.2, 22.5, 25.6, 27.5, 29.7, 47.8, 48.8,

56.5, 69.9, 83.5, 85.7, 85.8, 91.8, 96.4, 115.2, 117.0, 120.4, 122.5,

129.7, 131.0, 132.4, 140.8, 149.6, 154.1, 155.6, 156.3, 176.8; HRMS

(ESI-) calcd for C₂₆H₃₃N₆O₉S [M - H]^-605.2035, found

605.2049 (error 2.3 ppm).

N⁶-Cyclobutyl-2′,3′-O-isopropylideneadenosine (56). The title

compound was synthesized from 48 (400 mg, 1.4 mmol, 1.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (3:1

EtOAc/hexane) provided the product (250 mg, 49%) as a white

1

N⁶-Isobutyl-2′ ,3′ -O-isopropylidene-5′ -O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (150 mg, 0.21 mmol, 1.0 equiv) prepared above was converted

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) followed by preparative HPLC using method 2 and

conversion to the sodium salt using the general procedure for ion

exchange afforded the title compound (26 mg, 23%) as a white

solid: mp >200 °C (dec); R_f) 0.53 (4:1 EtOAc/MeOH); [R]_D²⁷

-25.6 (c 0.156, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.01 (d,

J ) 6.6 Hz, 6H), 1.98 (hept, J ) 6.6 Hz, 1H), 3.40 (br s, 2H),

4.31-4.33 (m, 1H), 4.37 (dd, J ) 10.8, 3.0 Hz, 1H), 4.40-4.43

(m, 2H), 4.70 (t, J ) 5.4 Hz, 1H), 6.08 (d, J ) 6.0 Hz, 1H),

6.75-6.79 (m, 2H), 7.28 (t, J ) 7.8 Hz, 1H), 7.94 (d, J ) 7.8 Hz,

1H), 8.20 (br s, 1H), 8.46 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 20.5, 29.7, 48.8, 69.5, 72.4, 76.1, 84.6, 89.2, 117.9, 119.2, 120.4,

120.6, 131.3, 134.4, 140.4, 149.9, 153.9, 156.3, 162.1, 175.2; HRMS

(ESI-) calcd for C₂₁H₂₅N₆O₈S [M - H]^-521.1460, found

521.1463 (error 0.6 ppm).

solid: mp 126-127 °C; R_f) 0.39 (EtOAc); H NMR (600 MHz,

CDCl₃) δ 1.37 (s, 3H), 1.64 (s, 3H), 1.77-1.82 (m, 2H), 1.96-2.03

(m, 2H), 2.45-2.51 (m, 2H), 3.77 (d, J ) 12.6 Hz, 1H), 3.96 (d,

J ) 12.6 Hz, 1H), 4.53 (s, 1H), 4.76 (br s, 1H), 5.11 (d, J ) 5.4

Hz, 1H), 5.19 (t, J ) 5.4 Hz, 1H), 5 83 (d, J ) 5.4 Hz, 1H), 7.77

(s, 1H), 8.31 (br s, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 15.3, 25.4,

27.8, 31.7, 45.9, 63.6, 81.9, 83.1, 86.2, 94.6, 114.1, 121.3, 139.7,

147.5, 152.9, 154.4; HRMS (ESI+) calcd for C₁₇H₂₄N₅O₄[M +

H]⁺362.1823, found 362.1846 (error 6.4 ppm).

N⁶-Cyclobutyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (66). Compound 56 (200 mg, 0.55 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (9:1 EtOAc/

hexanes) afforded the title compound (220 mg, 91%) as a viscous

1

oil: R_f) 0.53 (EtOAc); H NMR (600 MHz, CD₃OD) δ 1.38 (s,

3H), 1.60 (s, 3H), 1.77-1.82 (m, 2H), 2.05-2.11 (m, 2H),

2.41-2.46 (m, 2H), 4.26 (dd, J ) 10.8, 4.8 Hz, 1H), 4.33 (dd, J )

10.8, 4.5 Hz, 1H), 4.50-4.52 (m, 1H), 4.67 (br s, 1H), 5.12-5.13

(m, 1H), 5.39-5.41 (m, 1H), 6.23 (s, 1H), 8.21 (s, 1H), 8.23 (br s,

1H); ¹³C NMR (150 MHz, CD₃OD) δ 15.9, 25.5, 27.4, 31.9, 47.0,

69.9, 82.9, 85.5, 85.7, 91.7, 115.6, 120.5, 140.9, 149.5, 154.1, 155.0;

HRMS (ESI+) calcd for C₁₇H₂₅N₆O₆S [M + H]⁺441.1556, found

441.1558 (error 0.5 ppm).

N⁶-Cyclopropyl-2′,3′-O-isopropylideneadenosine (55). The title

compound was synthesized from 48 (176 mg, 0.61 mmol, 1.0 equiv)

and cyclopropylamine (85 µL, 1.22 mmol, 2.0 equiv) using the

general one-pot procedure for N⁶-amination and isopropylidene

protection. Puriﬁcation by ﬂash chromatography (50:50 EtOAc/

hexane) afforded the title compound (200 mg, 94%): R_f) 0.4

(EtOAc); [R]²_D⁰-230.3 (c 1.0, MeOH); ¹H NMR (600 MHz, CDCl₃)

δ 0.67 (br s, 2H), 0.95 (q, J ) 6.0 Hz, 2H), 1.37 (s, 3H), 1.63 (s,

3H), 3.06 (br s, 1H), 3.78 (d, J ) 12.6 Hz, 1H), 3.96 (d, J ) 13.2

Hz, 1H), 4.53 (s, 1H), 5.11 (d, J ) 5.4 Hz, 1H), 5.19 (t, J ) 5.4

Hz, 1H), 5.86 (d, J ) 5.4 Hz, 1H), 7.81 (br s, 1H), 8.40 (br s, 1H);

¹³C NMR (150 MHz, CDCl₃) δ 7.6, 23.9, 25.4, 27.8, 63.5, 81.9,

83.2, 86.3, 94.5, 114.2, 121.5, 140.0, 153.1, 156.2, 164.0; HRMS

(APCI-) calcd for C₁₆H₂₀N₅O₄[M + H]⁺346.1521, found

346.1551 (error 8.7).

N⁶-Cyclobutyl-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Sodium Salt (17). Compound 66 (110 mg, 0.25 mmol, 1.0 equiv)

was coupled to 33 using the general procedure for salicylation.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

afforded

N⁶-cyclobutyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (150 mg, 85%) as a viscous oil: R_f) 0.49 (EtOAc); ¹H NMR

(600 MHz, CD₃OD) δ 1.25 (t, J ) 7.2 Hz, 9H), 1.37 (s, 3H), 1.61

(s, 3H), 1.77-1.84 (m, 2H), 2.05-2.11 (m, 2H), 2.42-2.47 (m,

2H), 3.13 (q, J ) 7.2 Hz, 6H), 3.42 (s, 3H), 4.31-4.39 (m, 2H),

4.60 (s, 1H), 4.68 (br s, 1H), 5.16 (s, 2H), 5.22 (d, J ) 2.4 Hz,

1H), 5.39 (s, 1H), 6.25 (s, 1H), 6.97 (t, J ) 7.2 Hz, 1H), 7.12 (d,

J ) 8.4 Hz, 1H), 7.27 (t, J ) 7.2 Hz, 1H), 7.41 (d, J ) 7.2 Hz,

1H), 8.23 (br s, 1H), 8.46 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 9.2, 15.9, 25.6, 27.5, 31.9, 47.0, 47.8, 56.6, 69.9, 83.4, 85.72,

85.74, 91.8, 96.4, 115.2, 117.0, 120.2, 122.5, 129.7, 131.0, 132.5,

140.9, 149.8, 154.1, 155.0, 155.6, 176.7; HRMS (ESI-) calcd for

C₂₆H₃₁N₆O₉S [M - H]^-603.1879, found 603.1878 (error 0.2 ppm)

N⁶-Cyclopropyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (65). Compound 55 (200 mg, 0.58 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (50:50 EtOAc/

hexane) provided the product (190 mg, 76%): R_f) 0.5 (EtOAc);

[R]²_D⁰-57.9 (c 1.80, MeOH); ¹H NMR (600 MHz, CD₃OD) δ

0.66-0.68 (m, 2H), 0.89-0.93 (m, 2H), 1.41 (s, 3H), 1.63 (s, 3H),

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5363

N⁶-Cyclobutyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (120 mg, 0.17 mmol, 1.0 equiv) prepared above was converted

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) followed by preparative HPLC using method 1 and

conversion to the sodium salt using the general procedure for ion

exchange afforded the title compound (18 mg, 20%) as a white

solid (reported NMR data are for the TEA salt of the title

compound): mp >200 °C (dec); R_f) 0.55 (4:1 EtOAc/MeOH);

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) followed by preparative HPLC using method 1 and

conversion to the sodium salt using the general procedure for ion

exchange afforded the title compound (17 mg, 28%) as a white

solid (reported NMR data are for the TEA salt of the title

compound): mp >200 °C (dec); R_f) 0.53 (80:20 EtOAc/MeOH);

1

[R]²_D⁷-28.8 (c 0.163, MeOH); H NMR (600 MHz, CD₃OD) δ

1.25 (t, J ) 7.2 Hz, 9H), 1.60-1.72 (m, 4H), 1.80-1.86 (m, 2H),

2.07-2.12 (m, 2H), 3.09 (q, J ) 7.2 Hz, 6H), 4.30-4.32 (m, 1H),

4.37 (dd, J ) 10.8, 2.4 Hz, 1H), 4.40-4.43 (m, 2H), 4.52 (br s,

1H), 4.71 (t, J ) 5.4 Hz, 1H), 6.09 (d, J ) 5.4 Hz, 1H), 6.77 (ovlp

t, J ) 7.8 Hz, 1H), 6.79 (ovlp d, J ) 7.8 Hz, 1H), 7.29 (t, J ) 7.8

Hz, 1H), 7.94 (d, J ) 7.8 Hz, 1H), 8.22 (br s, 1H), 8.48 (s, 1H);

¹³C NMR (150 MHz, CD₃OD) δ 9.3, 24.7, 34.0, 47.9, 53.5, 69.6,

72.4, 76.1, 84.6, 89.1, 117.9, 119.3, 120.4, 120.6, 131.4, 134.3,

140.4, 149.9, 154.0, 155.6, 162.1, 175.0; HRMS (ESI-) calcd for

C₂₂H₂₅N₆O₈S [M - H]^-533.1460, found 533.1450 (1.9 ppm).

N⁶-Benzyl-2′,3′-O-isopropylideneadenosine (58). The title com-

pound was synthesized from 33 (400 mg, 1.4 mmol, 1.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (2:1

EtOAc/hexanes) afforded the title compound as a viscous oil (280

1

[R]²_D⁷-32.5 (c 0.117, MeOH); H NMR (600 MHz, CD₃OD) δ

1.14 (t, J ) 7.2 Hz, 9H), 1.78-1.83 (m, 2H), 2.06-2.12 (m, 2H),

2.42-2.47 (m, 2H), 2.81 (q, J ) 7.2 Hz, 6H), 4.30-4.32 (m, 1H),

4.37 (dd, J ) 10.8, 3.0 Hz, 1H), 4.40-4.43 (m, 2H), 4.62-4.71

(m, 2H), 6.08 (d, J ) 6.0 Hz, 1H), 6.76-6.80 (m, 2H), 7.29 (t, J

) 7.8 Hz, 1H), 7.94 (d, J ) 7.8 Hz, 1H), 8.20 (br s, 1H), 8.48 (s,

1H); ¹³C NMR (150 MHz, CD₃OD) δ 10.3, 15.9, 31.9, 47.1, 47.3,

69.6, 72.4, 76.1, 84.6, 89.1, 117.9, 119.2, 120.3, 120.6, 131.4, 134.3,

140.6, 150.2, 153.9, 155.0, 162.1, 175.0; HRMS (ESI-) calcd for

C₂₁H₂₃N₆O₈S [M - H]^-519.1304, found 519.1306 (0.4 ppm).

N⁶-Cyclopentyl-2′,3′-O-isopropylideneadenosine (57). The title

compound was synthesized from 48 (400 mg, 1.4 mmol, 1.0 equiv)

using the general one-pot procedure for N⁶-amination and isopro-

pylidene protection. Puriﬁcation by ﬂash chromatography (75:25

EtOAc/hexane) afforded the title compound (300 mg, 57%) as a

1

mg, 50%): R_f) 0.48 (EtOAc); H NMR (600 MHz, CD₃OD) δ

1.36 (s, 3H), 1.60 (s, 3H), 3.71 (dd, J ) 12.0, 3.9 Hz, 1H), 3.78

(dd, J ) 12.0, 3.6 Hz, 1H), 4.35-4.38 (m, 1H), 4.78 (br s, 2H),

5.03 (dd, J ) 6.0, 2.4 Hz, 1H), 5.26 (dd, J ) 6.0, 3.6 Hz, 1H),

6.13 (d, J ) 3.6 Hz, 1H), 7.21 (t, J ) 7.2 Hz, 1H), 7.28 (t, J ) 7.2

Hz, 2H), 7.35 (d, J ) 7.2 Hz, 1H), 8.24 (br s, 2H); ¹³C NMR (150

MHz, CD₃OD) δ 25.6, 27.6, 45.05, 63.6, 82.9, 85.2, 88.0, 92.9,

115.2, 121.1, 128.2, 128.5, 129.5, 140.2, 141.3, 149.3, 153.8, 156.1;

HRMS (ESI+) calcd for C₂₀H₂₄N₅O₄[M + H]⁺398.1823, found

398.1840 (error 4.3 ppm).

1

viscous oil: R_f) 0.35 (EtOAc); H NMR (600 MHz, CD₃OD) δ

1.33 (s, 3H), 1.54-1.66 (m, 7H), 1.72-1.78 (m, 2H), 2.01-2.08

(m, 2H), 3.71 (dd, J ) 12.0, 3.6 Hz, 1H), 3.78 (dd, J ) 12.0, 3.0

Hz, 1H), 4.34-4.36 (m, 1H), 4.47 (br s, 1H), 5.01 (dd, J ) 6.0,

1.8 Hz, 1H), 5.24 (dd, J ) 6.0, 3.8 Hz, 1H), 6.09 (d, J ) 3.6 Hz,

1H), 8.20 (br s, 1H), 8.22 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 24.7, 25.6, 27.6, 33.9, 53.4, 63.6, 82.9, 85.2, 87.9, 93.0, 115.1,

120.9, 140.9, 148.9, 153.8, 155.6; HRMS (ESI+) calcd for

C₁₈H₂₆N₅O₄[M + H]⁺376.1979, found 376.1982 (error 0.8 ppm).

N⁶-Cyclopentyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (67). Compound 57 (270 mg, 0.72 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (90:10 EtOAc/

hexanes) afforded the title compound (170 mg, 78%) as a viscous

N⁶-Benzyl-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (68).

Compound 58 (180 mg, 0.45 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (4:1 EtOAc/hexanes) afforded

the title compound (190 mg, 89%) as a viscous oil: R_f) 0.59

1

(EtOAc); H NMR (600 MHz, CD₃OD) δ 1.36 (s, 3H), 1.59 (s,

1

oil: R_f) 0.49 (EtOAc); H NMR (600 MHz, CD₃OD) δ 1.38 (s,

3H), 4.26 (dd, J ) 10.5, 4.8 Hz, 1H), 4.34 (dd, J ) 10.5, 4.8 Hz,

1H), 4.51 (td, J ) 4.8, 3.0 Hz, 1H), 4.78 (br s, 2H), 5.12 (dd, J )

6.0, 3.0 Hz, 1H), 5.39 (dd, J ) 6.0, 2.4 Hz, 1H), 6.22 (d, J ) 2.4

Hz, 1H), 7.21 (t, J ) 7.5 Hz, 1H), 7.28 (t, J ) 7.5 Hz, 2H), 7.35

(d, J ) 7.5 Hz, 1H), 8.19 (s, 1H), 8.26 (br s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 25.5, 27.4, 45.0, 69.9, 82.9, 85.4, 85.6, 91.7, 115.6,

120.7, 128.2, 128.5, 129.5, 140.1, 140.9, 149.5, 154.1, 156.0; HRMS

(ESI+) calcd for C₂₀H₂₅N₆O₆S [M + H]⁺477.1551, found

477.1554 (error 0.6 ppm).

3H), 1.58-1.71 (m, 7H), 1.77-1.83 (m, 2H), 2.05-2.11 (m, 2H),

4.26 (dd, J ) 10.8, 5.4 Hz, 1H), 4.33 (dd, J ) 10.8, 4.5 Hz, 1H),

4.50-4.52 (m, 2H), 5.13 (dd, J ) 6.3, 4.2 Hz, 1H), 5.41 (dd, J )

6.3, 3.0 Hz, 1H), 6.23 (d, J ) 3.0 Hz, 1H), 8.21 (s, 1H), 8.25 (br

s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.7, 25.5, 27.4, 33.9,

53.6, 69.9, 82.9, 85.5, 85.7, 91.7, 115.6, 120.6, 140.7, 149.3, 154.2,

155.7; HRMS (ESI-) calcd for C₁₈H₂₅N₆O₆S [M - H]^-453.1562,

found 453.1536 (error 5.7 ppm).

N⁶-Cyclopentyl-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Sodium Salt (18). Compound 67 (92 mg, 0.33 mmol, 1.0 equiv)

was coupled to 33 using the general procedure for salicylation.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

N⁶-Benzyl-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine So-

dium Salt (19). Compound 68 (130 mg, 0.27 mmol, 1.0 equiv) was

coupled to 33 using the general procedure for salicylation. Puriﬁca-

tion by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N) af-

afforded

N⁶-cyclopentyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

forded

N⁶-benzyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (148 mg, 62%) as a viscous oil: R_f) 0.64 (80:20 EtOAc/

MeOH); ¹H NMR (600 MHz, CD₃OD) δ 1.26 (t, J ) 7.2 Hz, 9H),

1.37 (s, 3H), 1.58-1.72 (m, 7H), 1.78-1.84 (m, 2H), 2.07-2.12

(m, 2H), 3.14 (q, J ) 7.2 Hz, 6H), 3.42 (s, 3H), 4.33 (dd, J )

11.1, 3.9 Hz, 1H), 4.37 (dd, J ) 11.1, 3.9 Hz, 1H), 4.37 (br s, 1H),

4.59-4.61 (m, 1H), 5.20 (s, 2H), 5.23 (d, J ) 5.4 Hz, 1H),

5.39-5.41 (m, 1H), 6.23 (d, J ) 3.6 Hz, 1H), 6.97 (t, J ) 7.2 Hz,

1H), 7.12 (d, J ) 7.2 Hz, 1H), 7.27 (t, J ) 7.2 Hz, 1H), 7.41 (d,

J ) 7.2 Hz, 1H), 8.24 (br s, 1H), 8.46 (s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 9.2, 24.7, 25.6, 27.5, 34.0, 47.8, 53.6, 56.6, 69.9,

83.5, 85.7, 85.8, 91.8, 96.4, 115.2, 117.0, 120.3, 122.5, 129.7, 131.0,

132.5, 133.9, 140.8, 149.5, 154.1, 155.6, 176.8; HRMS (ESI-)

calcd for C₂₇H₃₃N₆O₉S [M - H]^-617.2035, found 617.2043 (error

1.3 ppm).

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (140 mg, 70%) as a viscous oil: R_f) 0.55 (EtOAc); ¹H NMR

(600 MHz, CD₃OD) δ 1.24 (t, J ) 7.2 Hz, 1H), 1.37 (s, 3H), 1.62

(s, 3H), 3.12 (q, J ) 7.2 Hz, 6H), 3.41 (s, 3H), 4.34 (dd, J ) 10.8,

3.6 Hz, 1H), 4.37 (dd, J ) 10.8, 3.6 Hz, 1H), 4.59-4.62 (m, 1H),

4.80 (br s, 2H), 5.15 (s, 2H), 5.23 (dd, J ) 6.0, 1.8 Hz, 1H), 5.42

(dd, J ) 6.0, 3.0 Hz, 1H), 6.25 (d, J ) 3.0 Hz, 1H), 6.95 (t, J )

7.2 Hz, 1H), 7.11 (d, J ) 8.4 Hz, 1H), 7.22-7.27 (m, 2H), 7.30 (t,

J ) 7.5 Hz, 2H), 7.37 (d, J ) 7.8 Hz, 2H), 7.41 (d, J ) 7.8 Hz,

1H), 8.26 (br s, 1H), 8.47 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 9.2, 25.6, 27.5, 45.0, 47.8, 56.6, 69.9, 83.5, 85.7, 85.8, 91.9, 96.4,

115.2, 117.0, 120.5, 122.5, 128.2, 128.6, 129.5, 129.7, 131.0, 132.5,

140.3, 141.0, 150.0, 154.1, 155.6, 156.1, 176.7; HRMS (ESI-)

calcd for C₂₉H₃₁N₆O₉S [M - H]^-639.1879, found 639.1885 (error

0.9 ppm).

N⁶-Cyclopentyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (96 mg, 0.13 mmol, 1.0 equiv) prepared above was converted

N⁶-Benzyl-2′,3′-O-isopropylidene-5′-O-{N-[(2-methoxymethoxy)-

benzoyl]sulfamoyl}adenosine triethylammonium salt (130 mg, 0.18

mmol, 1.0 equiv) prepared above was converted to the title

5364 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

compound using the general procedure for TFA deprotection.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

followed by conversion to the sodium salt using the general

procedure for ion exchange afforded the title compound (44 mg,

42%) as a white solid: mp >200 °C (dec); R_f) 0.62 (4:1 EtOAc/

MeOH); [R]²_D⁷-28.4 (c 0.134, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 4.32 (dd, J ) 6.0, 3.0 Hz, 1H), 4.37 (dd, J ) 11.4, 3.6

Hz, 1H), 4.40-4.43 (m, 2H), 4.70, (t, J ) 5.4 Hz, 1H), 4.80 (br s,

2H), 6.10 (d, J ) 6.0 Hz, 1H), 6.74-6.79 (m, 2H), 7.23-7.29 (m,

2H), 7.31 (t, J ) 7.2 Hz, 2H), 7.38 (d, J ) 7.2 Hz, 2H), 7.93 (dd,

J ) 7.8, 1.8 Hz, 1H), 8.23 (s, 1H), 8.47 (br s, 1H); ¹³C NMR (150

MHz, CD₃OD) δ 45.1, 69.6, 72.3, 76.0, 84.5, 89.3, 117.9, 119.3,

120.5, 120.6, 128.2, 128.5, 129.5, 131.4, 134.4, 140.2, 140.6, 150.1,

153.9, 156.0, 162.0, 174.8; HRMS (ESI-) calcd for C₂₄H₂₃N₆O₈S

[M - H]^-555.1304, found 533.1304 (error 0 ppm).

Puriﬁcation by ﬂash chromatography (4:1 EtOAc/hexane) afforded

the title compound (230 mg, 48%) as a white solid: R_f) 0.5

(EtOAc); [R]²_D⁰-0.29 (c 2.0, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.36 (s, 3H), 1.56 (s, 3H), 4.17 (dd, J ) 10.2, 5.4 Hz,

1H), 4.30 (dd, J ) 10.2, 5.4 Hz, 1H), 4.38 (td, J ) 5.4, 3.0, Hz,

1H), 5.16 (dd, J ) 6.0, 3.0 Hz, 1H), 5.54 (d, J ) 6.0 Hz, 1H), 6.03

(s, 1H), 8.10 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 25.6, 27.5,

69.7, 83.1, 84.7, 86.4, 90.0, 115.6, 118.7, 146.6, 150.8, 153.2, 155.7;

MS (APCI+) calcd for C₁₃H₁₈N₉O₆S [M + H]⁺428.1, found 428.1.

8-Azido-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine Tri-

ethylammonium Salt (21). Compound 73 (427 mg, 1.05 mmol, 1.00

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (95:5:1 EtOAc/MeOH/Et₃N) afforded the title

compound (350 mg, 55%): R_f) 0.2 (1:9 MeOH/EtOAc); [R]_D²⁰

8-Bromo-2′,3′-O-isopropylideneadenosine (70). 8-Bromoadenos-

ine 69 (1.50 g, 4.33 mmol, 1.0 equiv) was converted to the title

compound using the general procedure for acetonide protection.

Puriﬁcation by ﬂash chromatography (4:1 EtOAc/hexanes) provided

the product (1.09 g, 65%) as a white solid: R_f) 0.3 (EtOAc); [R]_D²⁰

1

-21.6 (c 1.0, MeOH); H NMR (600 MHz, CD₃OD) δ 1.28 (t, J

) 2.4 Hz, 9H), 3.18 (q, J ) 7.2 Hz, 6H), 4.27 (q, J ) 5.4 Hz, 1H),

4.38 (dd, J ) 10.2, 6.6 Hz, 1H), 4.50 (dd, J ) 12.0, 5.4 Hz, 1H),

4.58 (t, J ) 5.4 Hz, 1H), 5.10 (t, J ) 5.4 Hz, 1H), 5.90 (d, J ) 5.4

Hz, 1H), 6.77-6.81 (m, 2H), 7.30 (t, J ) 7.8 Hz, 1H), 7.92 (d, J

) 7.8 Hz, 1H), 8.04 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.3,

48.0, 70.0, 72.3, 72.9, 83.9, 89.3, 118.0, 118.6, 119.3, 120.7, 131.3,

134.4, 147.0, 151.4, 152.9, 155.5, 162.0, 174.8. HRMS (ESI-)

calcd for C₁₇H₁₆N₉O₈S [M - H]^-506.0848, found 506.0849 (error

0.2 ppm).

1

-16.2 (c 1.25, DMSO); H NMR (600 MHz, CDCl₃) δ 1.36 (s,

3H), 1.66 (s, 3H), 3.78 (d, J ) 12.6 Hz, 1H), 3.95 (d, J ) 12.6 Hz,

1H), 4.51 (s, 1H), 5.06 (d, J ) 5.4 Hz, 1H), 5.26 (t, J ) 5.4 Hz,

1H), 5.94 (s, 2H, NH₂), 6.09 (d, J ) 5.4 Hz, 1H), 8.25 (s, 1H); ¹³

C

NMR (150 MHz, CDCl₃) δ 25.7, 28.0, 63.5, 81.8, 82.7, 86.0, 94.0,

114.3, 120.9, 127.4, 150.1, 152.7, 154.8; MS (APCI+) calcd for

C₁₃H₁₇BrN₅O₄[M + H]⁺386.0, found 386.1.

8-Amino-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine Tri-

ethylammonium Salt (22). Compound 21 (100 mg, 0.164 mmol,

1.0 equiv) was treated with Pd/C (20.0 mg) in MeOH (10 mL)

under a H₂(1 atm) for 2 h at room temperature. The mixture was

ﬁltered through Celite and concentrated. Puriﬁcation by ﬂash

chromatography (90:10:1 EtOAc/MeOH/Et₃N) afforded the title

compound (69 mg, 73%) as a white solid: R_f) 0.3 (80:20 EtOAc/

MeOH); [R]²_D⁰-19 (c 1.0, MeOH); ¹H NMR (600 MHz, CD₃OD)

δ 1.28 (t, J ) 7.2 Hz, 9H), 3.18 (q, J ) 7.2 Hz, 6H), 4.28-4.32

(m, 1H), 4.38-4.46 (m, 3H), 4.87-4.90 (ovlp m, 1H), 6.04 (d, J

) 7.2 Hz, 1H), 6.79-6.83 (m, 2H), 7.32 (t, J ) 7.8 Hz, 1H), 7.96

(d, J ) 7.8 Hz, 1H), 7.98 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 9.35, 48.0, 69.7, 71.9, 72.0, 84.6, 88.6, 117.8, 118.0, 119.4, 120.6,

131.5, 134.6, 150.4, 151.1, 153.5, 153.8, 162.1, 175.2; HRMS

(ESI-) calcd for C₁₇H₁₈N₇O₈S [M - H]^-480.0943, found

480.0960 (error 3.5 ppm).

2-Iodo-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine(82).Com-

pound 75^31,98,99(150 mg, 0.35 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash column chromatography (80:20 EtOAc/hexane)

provided the product (160 mg, 90%) as a viscous oil: R_f) 0.55

(3:1 EtOAc/hexanes); [R]²_D⁰-0.92 (c 1.3, CH₃OH); ¹H NMR (600

MHz, CD₃OD) δ 1.37 (s, 3H), 1.58 (s, 3H), 4.27 (dd, J ) 11.1,

5.4 Hz, 1H), 4.32 (dd, J ) 11.1, 5.4 Hz, 1H), 4.4-4.52 (m, 1H),

5.06-5.09 (m, 1H), 5.32 (d, J ) 6.0 Hz, 1H), 6.18 (s, 1H), 8.12

(s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 24.3, 26.2, 68.8, 81.7,

84.4, 84.9, 90.3, 114.4, 119.0, 119.6, 139.9, 149.4, 155.9; HRMS

(ESI+) calcd for C₁₃H₁₈IN₆O₆S [M + H]⁺513.0048, found

513.0056 (error 1.6 ppm).

8-Bromo-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (72).

Compound 70 (500 mg, 1.29 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash chromatography (7:3 EtOAc/hexane) provided

the product (510 mg, 85%) as a colorless oil: R_f) 0.6 (EtOAc);

1

[R]²_D⁰+1.0 (c 2.5, MeOH); H NMR (600 MHz, CD₃OD) δ 1.37

(s, 3H), 1.57 (s, 3H), 4.14 (dd, J ) 10.2, 7.2 Hz, 1H), 4.28 (dd, J

) 10.2, 6.0 Hz, 1H), 4.41-4.43 (m, 1H), 5.24 (dd, J ) 6.0, 3.0

Hz, 1H), 5.73 (d, J ) 6.0 Hz, 1H), 6.21 (s, 1H), 8.17 (s, 1H); ¹³

C

NMR (150 MHz, CD₃OD) δ 25.5, 27.5, 69.7, 83.5, 84.7, 86.9, 93.0,

115.6, 121.0, 128.6, 151.4, 154.2, 156.4; MS (APCI+) calcd for

C₁₃H₁₈BrN₆O₆S [M + H]⁺465.0, found 465.2.

8-Bromo-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine Tri-

ethylammonium Salt (20). Compound 72 (460 mg, 0.98 mmol, 1.00

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (9:1 EtOAc/MeOH) provided the product

(365 mg, 57%) as a white solid: R_f) 0.2 (1:4 MeOH/EtOAc);

1

[R]²_D⁰-5.7 (c 1.0, MeOH); H NMR (600 MHz, CD₃OD) δ 1.28

(t, J ) 7.2 Hz, 9H), 3.17 (q, J ) 7.2 Hz, 6H), 4.30 (td, J ) 6.0, 4.8

Hz, 1H), 4.35 (dd, J ) 10.2, 6.0 Hz, 1H), 4.53 (dd, J ) 10.2, 6.0

Hz, 1H), 4.63 (t, J ) 4.8 Hz, 1H), 5.39 (t, J ) 6.0 Hz, 1H), 6.03

(d, J ) 6.0 Hz, 1H), 6.76-6.81 (m, 2H), 7.30 (t, J ) 7.8 Hz, 1H),

7.90 (d, J ) 7.8 Hz, 1H), 8.03 (s, 1H); ¹³C NMR (150 MHz,

CD₃OD) δ 9.2, 47.9, 69.6, 72.3, 72.4, 84.2, 92.2, 117.8, 119.2,

120.7, 121.1, 129.1, 131.2, 134.2, 152.0, 153.8, 156.1, 161.9, 174.7;

HRMS (ESI-) calcd for C₁₇H₁₆BrN₆O₈S [M - H]^-542.9939,

found 542.9937 (error 0.4 ppm).

8-Azido-2′,3′-O-isopropylideneadenosine (71). Compound 70

(550 mg, 1.42 mmol, 1.0 equiv) and NaN₃(370 mg, 5.69 mmol, 4

equiv) were heated in DMF (10 mL) at 70 °C for 12 h. The reaction

mixture was concentrated in vacuo and the residue was puriﬁed by

ﬂash chromatography (1:1 EtOAc/hexane) to afford the title

compound (410 mg, 83%) as a white solid: R_f) 0.4 (EtOAc); [R]²_D⁰

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-iodoadenosine Triethy-

lammonium Salt (23). Compound 82 (112 mg, 0.21 mmol, 1.0

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N) afforded the

title compound (100 mg, 69%) as a white solid: R_f) 0.4 (1:9

MeOH/EtOAc); [R]²_D⁰-4.5 (c 0.32, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.27 (t, J ) 7.2 Hz, 9H), 3.18 (q, J ) 7.2 Hz, 6H),

4.31-4.33 (m, 1H), 4.38 (dd, J ) 11.4, 3.6 Hz, 1H), 4.41-4.43

(m, 2H), 4.65 (t, J ) 4.8 Hz, 1H), 6.01 (d, J ) 5.4 Hz, 1H),

6.74-6.82 (m, 2H), 7.28 (dt, J ) 8.4, 1.8 Hz, 1H), 7.93 (dd, J )

7.8, 1.8 Hz, 1H), 8.36 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ

9.2, 47.9, 69.8, 72.4, 76.1, 84.5, 89.5, 118.0, 119.4, 120.1, 120.7,

120.9, 131.4, 134.5, 140.8, 151.3, 157.1, 162.1, 174.9. HRMS

(ESI-) calcd for C₁₇H₁₆IN₆O₈S [M - H]^-590.9800, found

590.9784 (error 2.7 ppm).

1

-28 (c 1.2, MeOH); H NMR (600 MHz, CDCl₃) δ 1.35 (s, 3H),

1.63 (s, 3H), 3.75 (d, J ) 12.6 Hz, 1H), 3.92 (d, J ) 12.6 Hz, 1H),

4.63 (s, 1H), 5.05 (d, J ) 5.4 Hz, 1H), 5.19 (t, J ) 5.4 Hz, 1H),

5.82 (d, J ) 5.4 Hz, 1H), 8.19 (s, 1H); ¹³C NMR (150 MHz, CDCl₃)

δ 25.5, 27.8, 63.5, 81.7, 82.7, 85.9, 91.4, 114.1, 118.5, 145.4, 149.2,

151.7, 154.0; MS (APCI+) calcd for C₁₃H₁₇N₈O₄[M + H]⁺calcd

349.1, found 349.1.

8-Azido-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (73).

Compound 71 (390 mg, 1.12 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5365

2-Anilino-2′,3′-O-isopropylideneadenosine (76). Compound 76

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-(phenylethynyl)adenos-

ine Triethylammonium Salt (25). Compound 84 (35 mg, 0.072

mmol, 1.0 equiv) was converted to the title compound using the

general procedure for salicylation and TFA deprotection. Puriﬁca-

tion by ﬂash chromatography (95:5:1 EtOAc/MeOH/Et₃N) afforded

the product (25 mg, 52%) as a viscous oil: R_f) 0.35 (1:9 MeOH/

EtOAc); [R]²_D⁰+10.9 (c 0.110, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.25 (t, J ) 7.2 Hz, 9H), 3.14 (q, J ) 7.2 Hz, 6H),

4.32-4.36 (m, 1H), 4.38-4.49 (m, 3 H), 4.66 (t, J ) 5.4 Hz, 1H),

6.12 (d, J ) 5.4 Hz, 1H), 6.70-6.82 (m, 2H), 7.28 (t, J ) 7.2 Hz,

1H), 7.38-7.46 (m, 3H), 7.62 (d, J ) 8.4 Hz, 2H), 7.94 (d, J )

7.2 Hz, 1H), 7.59 (br s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.3,

48.0, 61.6, 69.6, 72.3, 76.5, 84.7, 86.2, 89.4, 117.9, 119.3, 120.8,

123.2, 129.8, 130.7, 131.5, 133.3, 134.4, 142.0, 147.8, 151.2, 157.2,

162.2, 175.2 (missing 1 Ar C); HRMS (ESI-) calcd for

C₂₅H₂₁N₆O₈S [M - H]^-565.1147, found 565.1140 (error 1.2 ppm).

2′,3′-O-Isopropylidene-2-phenyladenosine (78). Compound 78

was prepared as described.³²Results from ¹H NMR and HRMS

agreed with reported analytical data.³²Additional data for com-

pound 78: R_f) 0.5 (EtOAc); [R]²_D⁰-2.6 (c 0.41, MeOH); ¹³C NMR

(150 MHz, CD₃OD) δ 25.7, 27.7, 63.5, 83.2, 85.5, 88.7, 92.3, 115.4,

119.5, 129.3, 129.4, 131.0, 139.8, 142.1, 151.6, 157.4, 161.2.

2′,3′-O-Isopropylidene-2-phenyl-5′-O-(sulfamoyl)adenosine (85).

Compound 78 (40 mg, 0.10 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash column chromatography (9:1 EtOAc/hexane)

provided the product as a viscous oil (35 mg, 66%): R_f) 0.4

(EtOAc); [R]²_D⁰+40.2 (c 0.781, MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 1.40 (s, 3H), 1.63 (s, 3H), 4.27 (dd, J ) 10.8, 4.8 Hz,

1H), 4.34 (dd, J ) 10.8, 4.8 Hz, 1H), 4.50-4.60 (ovlp m, 1H),

5.18 (dd, J ) 5.4, 2.4 Hz, 1H), 5.50 (dd, J ) 6.0, 1.8 Hz, 1H),

6.27 (d, J ) 2.4 Hz, 1H), 7.40-7.50 (m, 3H), 8.12 (s, 1H),

8.20-8.35 (m, 2H); ¹³C NMR (150 MHz, CD₃OD) δ 24.7, 26.6,

68.5, 81.3, 84.0, 84.2, 90.2, 114.4, 117.8, 127.8, 128.1, 129.8, 137.7,

139.9, 149.9, 155.4, 159.7; HRMS ESI (+) calcd for C₁₉H₂₃N₆O₆S

[M + H]⁺463.1394, found 463.1397 (error 0.6 ppm).

1

was prepared as described.³²Results from H NMR and HRMS

agreed with reported analytical data.³²Additional data for com-

pound 76: R_f) 0.3 (1:19 MeOH/EtOAc); [R]²_D⁰-27.6 (c 0.31,

CH₃OH); ¹³C NMR (150 MHz, CD₃OD) δ 25.7, 27.6, 63.5, 8.1,

85.4, 88.5, 92.3, 115.2, 115.4, 120.8, 122.8, 129.6, 139.3, 142.3,

151.9, 157.6, 158.5.

2-Anilino-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenosine (83).

Compound 76 (90 mg, 0.23 mmol, 1.0 equiv) was converted to

the title compound using the general procedure for sulfamoylation.

Puriﬁcation by ﬂash column chromatography (1:19 MeOH/EtOAc)

provided the product (40 mg, 37%) as a viscous oil: R_f) 0.55

(1:19 MeOH/EtOAc); [R]²_D⁰+35.9 (c 0.472, MeOH); ¹H NMR (600

MHz, CD₃OD) δ 1.39 (s, 3H), 1.59 (s, 3H), 4.17 (dd, J ) 10.2,

5.4 Hz, 1H), 4.26 (dd, J ) 10.8, 4.8 Hz, 1H), 4.44-4.50 (m, 1H),

5.02 (dd, J ) 6.0, 2.4 Hz, 1H), 5.47 (dd, J ) 6.0, 2.4 Hz, 1H),

6.12 (d, J ) 2.4 Hz 1H), 6.96 (t, J ) 7.8 Hz), 7.27 (t, J ) 7.8 Hz,

2H), 7.64 (d, J ) 7.8 Hz, 2H), 7.93 (s, 1H); ¹³C NMR (150 MHz,

CD₃OD) δ 25.6, 27.5, 70.1, 83.1, 85.4, 85.9, 92.1, 115.4, 115.5,

121.2, 123.0, 129.7, 139.1, 142.2, 151.9, 157.5, 158.6; HRMS

(ESI+) calcd for C₁₉H₂₄N₇O₆S [M + H]⁺478.1503, found

478.1497 (error 1.3 ppm).

2-Anilino-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenosine Tri-

ethylammonium Salt (24). Compound 83 (40 mg, 0.083 mmol, 1.0

equiv) was converted to the title compound using the general

procedure for salicylation and TFA deprotection. Puriﬁcation by

ﬂash chromatography (1:4 MeOH/EtOAc) afforded the title com-

pound (24.4 mg, 45%) as a viscous oil: R_f) 0.7 (75:25:1 EtOAc/

1

MeOH/Et₃N); [R]²_D⁰+6.5 (c 0.20, MeOH); H NMR (600 MHz,

CD₃OD) δ 1.25 (t, J ) 7.2 Hz, 9H), 3.14 (q, J ) 7.2 Hz, 6H),

4.28-4.32 (m, 1H), 4.35 (dd, J ) 11.4, 3.6 Hz, 1H), 4.41 (dd, J )

4.8, 3.0 Hz, 1H), 4.43 (dd, J ) 11.4, 3.6 Hz, 1H), 4.78 (t, J ) 5.4

Hz, 1H), 6.10 (d, J ) 6.0 Hz, 1H), 6.74-6.84 (m, 2H), 6.92 (t, J

) 7.2 Hz, 1H), 7.24-7.32 (m, 3H), 7.68 (d, J ) 7.8 Hz, 2H), 7.94

(dd, J ) 7.8, 1.8 Hz, 1H), 8.18 (s, 1H); ¹³C NMR (150 MHz,

CD₃OD) δ 9.3, 48.0, 68.9, 72.6, 75.6, 84.4, 89.5, 115.2, 117.9,

119.4, 120.3, 120.8, 122.5, 129.7, 131.5, 134.4, 138.9, 142.4, 152.9,

157.4, 158.5, 162.2, 175.5; HRMS (ESI-) calcd for C₂₃H₂₂N₇O₈S

[M - H]^-556.1256, found 556.1252 (0.7 ppm error).

5′-O-[N-(2-Hydroxybenzoyl)sulfamoyl]-2-phenyladenosine Tri-

ethylammonium Salt (26). Compound 85 (32 mg, 0.068 mmol, 1.0

equiv) was coupled to compound 33 using the general procedure

for salicylation. Puriﬁcation by ﬂash chromatography (90:10:1

EtOAc/MeOH/Et₃N) afforded 2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}-2-phenyladenosine triethy-

2′,3′-O-Isopropylidene-2-(phenylethynyl)adenosine (77). A solu-

tion of 75 (100 mg, 0.23 mmol, 1.0 equiv), PdCl₂(PPh₃)₂(16.2 mg,

23.1 µmol, 0.1 equiv), CuI (8.8 mg, 46.2 µmmol, 0.2 equiv), Et₃N

(68 µL, 0.48 mmol, 2.1 equiv) in DMF (0.4 mL) was heated at 80

°C for 16 h. The mixture was cooled to room temperature, diluted

with CHCl₃(10 mL), ﬁltered through Celite, and the ﬁltrate was

concentrated. Puriﬁcation by ﬂash chromatography (95:5 EtOAc/

MeOH) afforded the title compound (53 mg, 57%) as a viscous

1

lammonium salt (39.3 mg, 87%): R_f) 0.38 (EtOAc); H NMR

(600 MHz, CD₃OD) δ 1.23 (t, J ) 7.2 Hz, 9H), 1.40 (s, 3H), 1.65

(s, 3H), 3.12 (q, J ) 7.2 Hz, 1H), 3.40 (s, 3H), 4.33-4.44 (m,

2H), 4.61-4.67 (m, 1H), 5.13 (s, 2H), 5.28 (dd, J ) 6.0, 2.4 Hz,

1H), 5.53 (dd, J ) 6.0, 3.0 Hz, 1H), 6.37 (d, J ) 3.0 Hz, 1H), 6.94

(t, J ) 7.2 Hz, 1H), 7.10 (d, J ) 9.0 Hz, 1H), 7.25 (t, J ) 7.8 Hz,

1H), 7.37 (d, J ) 7.8 Hz, 1H), 7.40-7.46 (m, 3H), 8.37 (d, J )

7.8 Hz, 2H), 8.44 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.2,

25.7, 27.7, 47.9, 56.6, 70.1, 83.5, 85.8, 85.9, 91.8, 96.4, 106.4,

115.4, 117.0, 119.1, 122.6, 129.3, 129.8, 130.9, 131.1, 132.5, 139.7,

141.7, 151.8, 155.7, 157.3, 161.1, 176.7; HRMS (ESI+) calcd for

C₂₈H₃₁N₆O₉S [M - H]^-625.1722, found 627.1720 (error 0.3 ppm).

2′,3′-O-Isopropylidene-5′-O-{N-[(2-methoxymethoxy)benzoyl-

]sulfamoyl}-2-phenyladenosine triethylammonium salt (23 mg,

0.032 mmol, 1.0 equiv) prepared above was converted to the title

compound using the general procedure for TFA deprotection.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

afforded the title compound (16.6 mg, 83%): R_f) 0.2 (3:7 MeOH/

EtOAc); [R]²_D⁰+7.4 (c 0.56, MeOH); ¹H NMR (600 MHz, CD₃OD)

δ 1.25 (t, J ) 7.2 Hz, 9H), 3.14 (q, J ) 7.2 Hz, 6H), 4.32-4.37

(m, 1H), 4.39 (dd, J ) 11.4, 3.6 Hz, 1H), 4.45 (dd, J ) 11.4, 3.6

Hz, 1H,), 4.49 (t, J ) 4.2 Hz, 1H), 4.82-4.86 (m, 1H), 6.22 (d, J

) 5.4 Hz, 1H), 6.74-6.84 (m, 2H), 7.28 (dt, J ) 8.4, 1.8 Hz, 1H),

7.38-7.46 (m, 3H), 7.94 (dd, J ) 7.8, 1.8 Hz, 1H), 8.36 (dd, J )

8.4, 1.8 Hz, 2H), 8.47 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ

9.3, 48.0, 69.8, 72.6, 75.9, 84.5, 89.3, 117.9, 119.1, 119.4, 120.7,

129.3, 129.4, 130.9, 131.4, 134.4, 139.8, 141.5, 152.3, 157.2, 161.1,

162.1, 175.1; HRMS (ESI+) calcd for C₂₃H₂₃N₆O₈S [M + H]⁺

543.1293, found 543.1291 (error 0.4 ppm).

1

oil: R_f) 0.3 (EtOAc); H NMR (600 MHz, CD₃OD) δ 1.40 (s,

3H), 1.66 (s, 3H), 3.83 (d, J ) 12.6 Hz, 1H), 3.98 (d, J ) 12.6 Hz,

1H), 4.51 (s, 1H), 5.12 (d, J ) 5.4 Hz, 1H), 5.21 (t, J ) 5.4 Hz,

1H), 5.99 (d, J ) 5.4 Hz, 1H), 7.35-7.45 (m, 3H), 7.66 (d, J )

7.8 Hz, 2H), 8.11 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 29.2,

31.4, 67.1, 85.6, 87.5, 90.2, 90.7, 91.6, 97.3, 118.4, 123.7, 125.5,

132.5, 133.6, 136.5, 145.2, 150.6, 159.7 (missing 1 Ar C); HRMS

(ESI+) calcd for C₂₁H₂₂N₅O₄S [M + H]⁺408.1666, found

408.1635 (error 7.6 ppm).

2′,3′-O-Isopropylidene-2-phenylethynyl-5′-O-(sulfamoyl)adenos-

ine (84). Compound 77 (50 mg, 0.12 mmol, 1.0 equiv) was

converted to the title compound using the general procedure for

sulfamoylation. Puriﬁcation by ﬂash chromatography (1:49 MeOH/

EtOAc) provided the product (39 mg, 66%) as a viscous oil: R_f)

0.5 (1:19 MeOH/EtOAc); [R]²_D⁰-15.0 (c 0.162, MeOH); ¹H NMR

(600 MHz, CD₃OD) δ 1.41 (s, 3H), 1.63 (s, 3H), 4.31 (dd, J )

10.8, 5.4 Hz, 1H), 4.36 (dd, J ) 11.4, 4.8 Hz, 1H), 4.52-4.58 (m,

1H), 5.14 (dd, J ) 6.0, 3.0 Hz, 1H), 5.40 (dd, J ) 6.0, 3.0 Hz,

1H), 6.26 (d, J ) 3.0 Hz, 1H), 7.38-7.48 (m, 3H), 7.61 (d, J )

7.8 Hz, 2H), 8.33 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 25.6,

27.6, 70.1, 83.0, 85.6, 85.7, 86.3, 89.3, 91.7, 115.9, 123.1, 129.8,

130.0, 130.7, 133.4, 142.3, 147.9, 157.3 (missing 1 Ar C); HRMS

(ESI+) calcd for C₂₁H₂₃N₆O₆S [M + H]⁺487.1394, found

487.1395 (error 0.2 ppm).

5366 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

2-(Biphen-2-yl)-2′,3′-O-isopropylideneadenosine (79). To a solu-

tion of compound 75 (150 mg, 0.346 mmol, 1.0 equiv) in 1,4-

dioxane (5.0 mL) were added 2-biphenylboronic acid (137 mg, 0.69

mmol, 2.0 equiv), Pd(OAc)₂(15.5 mg, 0.0692 mmol, 0.2 equiv),

2-(biphenyl)dicyclohexylphosphine (36.4 mg, 0.104 mmol, 0.30

equiv), K₃PO₄(183 mg, 0.87 mmol, 2.5 equiv), and the mixture

was heated at 100 °C for 12 h. After cooling to room temperature,

the mixture was diluted with MeOH (20 mL), ﬁltered, and

concentrated. Puriﬁcation by ﬂash chromatography (gradient from

80-100% EtOAc/hexane) afforded the title compound (92 mg,

58%) as a white solid: R_f) 0.30 (80:20 EtOAc/hexane); ¹H NMR

(600 MHz, CD₃OD) δ 1.40 (s, 3H), 1.58 (s, 3H), 3.52 (dd, J )

12.0, 4.2 Hz, 1H), 3.56 (dd, J ) 12.0, 4.2 Hz, 1H), 4.24-4.26 (m,

1H), 4.45 (dd, J ) 6.0, 4.2 Hz, 1H), 4.71 (dd, J ) 6.0, 1.8 Hz,

1H), 5.89 (d, J ) 4.2 Hz, 1H), 7.18-7.26 (m, 5H), 7.45-7.49 (m,

2H), 7.53 (t, J ) 7.8 Hz, 1H), 7.71 (d, J ) 7.8 Hz, 1H), 8.17 (s,

1H); ¹³C NMR (150 MHz, CD₃OD) δ 26.1, 27.8, 63.6, 83.1, 84.2,

87.7, 93.4, 115.0, 119.3, 127.7, 128.1, 128.9, 130.2, 130.3, 131.4,

131.5, 139.8, 142.5, 142.6, 143.1, 150.1, 157.3, 163.7; HRMS

(ESI+) calcd for C₂₅H₂₆N₅O₄[M + H]⁺460.1979, found 460.2005

(error 5.7 ppm).

2-(Biphen-2-yl)-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (86). Sulfamoyl chloride (78 mg, 0.68 mmol, 4.0 equiv) was

added to a solution of 79 (78 mg, 0.17 mmol, 1.0 equiv) in DMA

(2.5 mL) at 0 °C. The solution was stirred for 4 h at 0 °C and then

partitioned between EtOAc (40 mL) and H₂O (40 mL). The organic

layer was separated, washed with H₂O (3 × 40 mL), saturated

aqueous NaCl (40 mL), dried (Na₂SO₄), and concentrated to afford

the title compound (87 mg, 96%) as a white solid: R_f) 0.29 (80:

20 EtOAc/benzene); ¹H NMR (600 MHz, CD₃OD) δ 1.37 (s, 3H),

1.53 (s, 3H), 4.06 (dd, J ) 10.8, 6.6 Hz, 1H), 4.12 (dd, J ) 10.8,

5.4 Hz, 1H), 4.36-4.40 (m, 1H), 4.61 (dd, J ) 5.4, 2.4 Hz, 1H),

4.69 (dd, J ) 6.0, 1.8 Hz, 1H), 5.97 (d, J ) 2.4 Hz, 1H), 7.17-7.24

(m, 5H), 7.44-7.46 (m, 2H), 7.50 (t, J ) 7.8 Hz, 1H), 7.72 (d, J

) 7.2 Hz, 1H), 8.13 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ

26.3, 27.6, 69.7, 83.3, 84.7, 85.5, 92.5, 115.3, 118.9, 127.7, 128.3,

129.1, 130.06, 130.14, 131.6, 131.7, 140.0, 142.3, 142.4, 143.3,

150.3, 157.2, 163.7; HRMS (ESI+) calcd for C₂₅H₂₇N₆O₆S [M +

H]⁺539.1707, found 539.1716 (error 1.7 ppm).

2-(Biphen-2-yl)-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Sodium Salt (27). Compound 86 (74 mg, 0.14 mmol, 1.0 equiv)

was coupled to compound 33 using the general procedure for

salicylation. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) afforded 2-(biphen-2-yl)-2′,3′-O-isopropylidene-5′-

O-{N-[(2-methoxymethoxy)benzoyl]sulfamoyl}adenosine triethy-

lammonium salt (87 mg, 79%): R_f) 0.56 (90:10 EtOAc/MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.19 (t, J ) 7.2 Hz, 9H), 1.36 (s,

3H), 1.54 (s, 3H), 3.07 (q, J ) 7.2 Hz, 6H), 3.39 (s, 3H), 4.20 (dd,

J ) 10.8, 4.8 Hz, 1H), 4.23 (dd, J ) 10.8, 4.8 Hz, 1H), 4.49-4.53

(m, 1H), 4.84-4.87 (m, 1H), 4.88-4.94 (ovlp m, 1H), 5.11-5.13

(m, 2H), 6.00 (d, J ) 3.0 Hz, 1H), 6.96 (t, J ) 7.8 Hz, 1H), 7.10

(d, J ) 8.4 Hz, 1H), 7.16-7.23 (m, 5H), 7.27 (t, J ) 7.8 Hz, 1H),

7.37 (d, J ) 7.8 Hz, 1H), 7.41-7.44 (m, 2H), 7.48 (t, J ) 7.8 Hz,

1H), 7.72 (d, J ) 7.8 Hz, 1H), 8.34 (s, 1H); ¹³C NMR (150 MHz,

CD₃OD) δ 9.1, 26.2, 27.7, 47.6, 56.6, 69.8, 83.4, 85.0, 85.5, 92.0,

96.2, 115.1, 116.9, 118.6, 122.5, 127.6, 128.2, 129.0, 129.7, 130.0,

131.0, 131.49, 131.52, 132.3, 140.1, 142.1, 142.6, 143.2, 150.7,

155.6, 157.0, 163.7, 176.6 (missing 1 Ar C); HRMS (ESI-) calcd

for C₃₄H₃₃N₆O₉S [M - H]^-701.2035, found 701.2015 (error 2.9

ppm).

Hz, 1H), 7.67 (d, J ) 7.8 Hz, 1H), 7.93 (d, J ) 7.2 Hz, 1H), 8.37

(s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 69.4, 71.7, 75.5, 84.0,

90.2, 117.9, 118.6, 119.3, 120.6, 127.7, 128.1, 129.0, 130.1, 131.2,

131.3, 131.4, 140.2, 141.5, 142.7, 143.0, 150.8, 156.8, 162.1, 163.7,

175.2 (missing 2 Ar C); HRMS (ESI-) calcd for C₂₉H₂₅N₆O₈S

[M - H]^-617.1460, found 617.1463 (error 0.5 ppm).

2-(Biphen-3-yl)-2′,3′-O-isopropylideneadenosine (80). To a solu-

tion of compound 75 (300 mg, 0.69 mmol, 1.0 equiv) in 1,4-dioxane

(10 mL) were added 3-biphenylboronic acid (206 mg, 1.04 mmol,

1.5 equiv), Pd(OAc)₂(15.5 mg, 0.0692 mmol, 0.2 equiv), 2-(bi-

phenyl)dicyclohexylphosphine (36.4 mg, 0.104 mmol, 0.30 equiv),

K₃PO₄(294 mg, 1.38 mmol, 2.0 equiv), and the reaction was heated

at 100 °C for 18 h. After cooling to room temperature, the mixture

was diluted with EtOAc (50 mL), ﬁltered, and concentrated.

Puriﬁcation by ﬂash chromatography (gradient from 80-100%

EtOAc/hexane) afforded the title compound (215 mg, 68%) as a

white solid contaminated with approximately 10 mol% of 75. The

solid was recrystallized from EtOAc (15 mL) by reﬂuxing for 15

min. The solution was ﬁltered while hot and the ﬁltrate cooled to

room temperature, then placed at -20 °C for 8 h. The title

compound was isolated as a white crystalline solid (116 mg, 36%)

from the ﬁrst crop: R_f) 0.20 (80:20 EtOAc/hexane); ¹H NMR

(600 MHz, DMSO-d₆) δ 1.36 (s, 3H), 1.58 (s, 3H), 3.51-3.59 (m,

2H), 4.22 (td, J ) 5.4, 2.4 Hz, 1H), 5.02 (t, J ) 6.0 Hz, 1H), 5.10

(dd, J ) 6.0, 2.4 Hz, 1H), 5.56 (dd, J ) 6.0, 2.4 Hz, 1H), 6.27 (d,

J ) 2.4 Hz, 1H), 7.38-7.44 (m, 3H), 7.51 (t, J ) 7.2 Hz, 2H),

7.58 (t, J ) 7.2 Hz, 1H), 7.73-7.76 (m, 3H), 8.34-8.35 (m, 2H),

8.66 (s, 1H); ¹³C NMR (150 MHz, DMSO-d₆) δ 25.9, 27.7, 62.3,

82.3, 84.1, 87.6, 90.0, 113.7, 119.0, 126.8, 126.9, 127.39, 127.41,

128.3, 128.7, 129.7, 139.6, 140.89, 140.90, 141.1, 150.5, 156.6,

158; HRMS (APCI+) calcd for C₂₅H₂₆N₅O₄[M + H]⁺460.1979,

found 460.2025 (error 10.0 ppm).

2-(Biphen-3-yl)-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (87). Sulfamoyl chloride (78 mg, 0.68 mmol, 4.0 equiv) was

added to a solution of 80 (78 mg, 0.17 mmol, 1.0 equiv) in DMA

(2.5 mL) at 0 °C. The solution was stirred 4 h at 0 °C and then

partitioned between EtOAc (40 mL) and H₂O (40 mL). The organic

layer was separated, washed with H₂O (3 × 40 mL), saturated

aqueous NaCl (40 mL), dried (Na₂SO₄), and concentrated to afford

the title compound (87 mg, 96%) as a white solid: R_f) 0.40 (80:

20 EtOAc/benzene); ¹H NMR (600 MHz, CD₃OD) δ 1.38 (s, 3H),

1.60 (s, 3H), 4.14 (dd, J ) 10.8, 6.6 Hz, 1H), 4.24 (dd, J ) 10.8,

5.4 Hz, 1H), 4.45 (td, J ) 6.0, 3.0 Hz, 1H), 5.19 (dd, J ) 6.0, 3.6

Hz, 1H), 5.61 (dd, J ) 6.0, 1.8 Hz, 1H), 6.36 (d, J ) 1.8 Hz, 1H),

7.41 (t, J ) 7.8 Hz, 1H), 7.47 (br s, 2H), 7.51 (t, J ) 7.8 Hz, 2H),

7.56 (s, 2H), 7.59 (t, J ) 7.8 Hz, 1H), 7.73 (ovlp d, J ) 7.8 Hz,

2H), 7.75 (ovlp d, J ) 7.8 Hz, 1H), 8.33 (ovlp s, 1H), 8.34 (ovlp

d, J ) 7.8 Hz, 1H), 8.65 (s, 1H); ¹³C NMR (150 MHz, CD₃OD)

δ 25.1, 26.9, 68.1, 81.2, 83.4, 83.8, 88.9, 113.5, 118.3, 126.1,

126.71, 126.74, 127.6, 128.1, 129.0, 129.1, 140.23, 140.24, 140.3,

149.7, 156.0, 157.9 (missing 1 Ar C); HRMS (APCI+) calcd for

C₂₅H₂₇N₆O₆S [M + H]⁺539.1707, found 539.1706 (error 0.2 ppm).

2-(Biphen-3-yl)-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Sodium Salt (28). Compound 87 (87.4 mg, 0.162 mmol, 1.0

equiv) was coupled to compound 33 using the general procedure

for salicylation. Puriﬁcation by ﬂash chromatography (90:10:1

EtOAc/MeOH/Et₃N) afforded 2-(biphen-3-yl)-2′,3′-O-isopropyl-

idene-5′-O-{N-[(2-methoxymethoxy)benzoyl]sulfamoyl}adenosine tri-

ethylammonium salt (102 mg, 78%): R_f) 0.46 (95:5 EtOAc/MeOH);

¹H NMR (600 MHz, CD₃OD) δ 1.18 (t, J ) 7.2 Hz, 9H), 1.39 (s,

3H), 1.63 (s, 3H), 3.05 (q, J ) 7.2 Hz, 6H), 3.37 (s, 3H), 4.32-4.37

(m, 2H), 4.62-4.65 (m, 1H), 5.10 (s, 2H), 5.26 (dd, J ) 6.0, 1.8 Hz,

1H), 5.55 (dd, J ) 6.0, 2.4 Hz, 1H), 6.36 (d, J ) 2.4 Hz, 1H), 6.91 (t,

J ) 7.8 Hz, 1H), 7.07 (d, J ) 7.8 Hz, 1H), 7.23 (t, J ) 7.8 Hz, 1H),

7.32 (ovlp d, J ) 7.8 Hz, 1H), 7.34 (ovlp t, J ) 7.8 Hz, 1H), 7.44 (t,

J ) 7.8 Hz, 2H), 7.51 (t, J ) 7.8 Hz, 1H), 7.66 (ovlp d, J ) 7.8 Hz,

1H), 7.69 (ovlp d, J ) 7.8 Hz, 2H), 8.35 (d, J ) 7.8 Hz, 1H), 8.41 (s,

1H), 8.67 (s, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 9.1, 25.7, 27.6,

47.8, 56.5, 70.0, 83.5, 85.8, 86.0, 91.9, 96.3, 115.2, 116.9, 119.1, 122.5,

127.9, 128.0, 128.1, 128.5, 129.4, 129.6, 129.90, 129.92, 131.0, 132.4,

140.2, 141.7, 142.3, 142.4, 151.7, 155.6, 157.2, 160.7, 176.6; HRMS

2-(Biphen-2-yl)-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (87 mg, 0.11 mmol, 1.0 equiv) prepared above was converted

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N), followed by conversion to the sodium salt using the

general procedure for ion exchange afforded the title compound

1

(56 mg, 80%): R_f) 0.35 (90:10 EtOAc/MeOH); H NMR (600

MHz, CD₃OD) δ 4.22-4.29 (m, 4H), 4.36 (dd, J ) 10.8, 3.0 Hz,

1H), 5.82 (d, J ) 4.2 Hz, 1H), 6.76-6.81 (m, 2H), 7.15-7.21 (m,

5H), 7.30 (t, J ) 7.2 Hz, 1H), 7.41-7.45 (m, 2H), 7.48 (t, J ) 7.2

Inhibition of Siderophore Biosynthesis

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5367

1

(ESI-) calcd for C₃₄H₃₃N₆O₉S [M - H]^-701.2035, found 701.2029

(error 0.9 ppm).

R_f) 0.50 (95:5 EtOAc/MeOH); H NMR (600 MHz, DMSO-d₆)

δ 1.33 (s, 3H), 1.60 (s, 3H), 3.33 (s, 3H), 4.15 (d, J ) 4.2 Hz, 2H),

4.49 (td, J ) 4.2, 1.8 Hz, 1H), 5.07 (s, 2H), 5.20 (dd, J ) 6.0, 1.8

Hz, 1H), 5.48 (dd, J ) 6.0, 3.0 Hz, 1H), 6.29 (d, J ) 3.0 Hz, 1H),

6.89 (t, J ) 7.8 Hz, 1H), 6.99 (d, J ) 7.8 Hz, 1H), 7.18 (t, J ) 7.8

Hz, 1H), 7.32 (d, J ) 7.8 Hz, 1H), 7.37-7.42 (m, 3H), 7.49 (t, J

) 7.8 Hz, 2H), 7.73 (d, J ) 7.8 Hz, 2H), 7.78 (d, J ) 8.4 Hz, 2H),

8.45 (ovlp d, J ) 8.4 Hz, 2H), 8.46 (ovlp s, 1H); ¹³C NMR (150

MHz, DMSO-d₆) δ 25.2, 27.1, 55.5, 67.2, 81.7, 83.4, 83.8, 89.0,

95.0, 113.2, 116.8, 118.0, 121.2, 126.5, 126.7, 127.7, 128.3, 128.7,

128.8, 129.0, 133.1, 137.3, 139.7, 140.1, 141.1, 150.1, 153.8, 156.0,

157.8, 171.6; HRMS (ESI-) calcd for C₃₄H₃₃N₆O₉S [M - H]^-

701.2035, found 701.1984 (error 7.3 ppm).

2-(Biphen-4-yl)-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine cesium salt (42 mg,

0.059 mmol, 1.0 equiv) prepared above was converted to the title

compound using the general procedure for TFA deprotection.

Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/MeOH/Et₃N)

followed by conversion to the sodium salt using the general

procedure for ion exchange afforded the title compound (36 mg,

80%): R_f) 0.40 (90:10 EtOAc/MeOH); ¹H NMR (600 MHz,

CD₃OD) δ 4.35-4.37 (m, 1H), 4.41 (dd, J ) 10.8, 3.0 Hz, 1H),

4.47-4.52 (m, 2H), 4.88 (ovlp t, J ) 5.4 Hz, 1H), 6.22 (d, J ) 5.4

Hz, 1H), 6.75 (t, J ) 7.8 Hz, 1H), 6.78 (d, J ) 7.8 Hz, 1H), 7.27

(t, J ) 7.8 Hz, 1H), 7.36 (t, J ) 7.8 Hz, 1H), 7.46 (t, J ) 7.8 Hz,

2H), 7.68 (d, J ) 7.8 Hz, 2H), 7.70 (d, J ) 7.8 Hz, 2H), 7.94 (d,

J ) 7.8 Hz, 1H), 8.46 (ovlp d, J ) 7.8 Hz, 2H), 8.47 (ovlp s, 1H);

¹³C NMR (150 MHz, CD₃OD) δ 69.9, 72.7, 75.8, 84.5, 89.6, 118.0,

119.3, 119.4, 120.8, 127.9, 128.2, 128.7, 130.00, 130.03, 131.6,

134.5, 138.9, 141.7, 142.1, 143.9, 152.4, 157.3, 160.9, 162.2, 175.4;

HRMS (ESI-) calcd for C₂₉H₂₅N₆O₈S [M - H]^-617.1460, found

617.14541 (error 1.0 ppm).

Enzyme Kinetic Studies. ATP-PP_iExchange Assay for MbtA,

FadD17, FadD19, FadD26, and FadD28. MbtA was expressed in

E. coli and puriﬁed as described.²¹FadD17, FadD19, FadD26, and

FadD28 were cloned and expressed in E. coli and puriﬁed as

described.³⁶MbtA concentration was determined by active site

titration with 6 as described.²¹The inhibition assays were performed

in duplicate as described.²¹In brief, the reaction was initiated by

adding 10 µL of [³²P]PP_iwith 7 nM MbtA in 90 µL of reaction

buffer (278 µM salicylic acid, 11.1 mM ATP, 1.11 mM PP_i, 83.3

mM Tris-HCl, pH 7.5, 11.1 mM MgCl₂, 2.22 mM DTT) at 37 °C

in the presence of eight different concentrations of the inhibitor.

The reaction was terminated by the addition of 200 µL of quenching

buffer (350 mM HClO₄, 100 mM PP_i, 1.8% w/v activated charcoal).

The charcoal was pelleted by centrifugation and washed once with

500 µL of H₂O and analyzed by liquid scintillation counting as

described. The K_I^appvalues were calculated using either the

Hill-Morrison (eq 1) or Morrison (eq 2) equations as described.²¹

2-(Biphen-3-yl)-2′,3′-O-isopropylidene-5′-O-{N-[(2-

methoxymethoxy)benzoyl]sulfamoyl}adenosine triethylammonium

salt (86 mg, 0.107 mmol, 1.0 equiv) prepared above was converted

to the title compound using the general procedure for TFA

deprotection. Puriﬁcation by ﬂash chromatography (90:10:1 EtOAc/

MeOH/Et₃N) followed by conversion to the sodium salt using the

general procedure for ion-exchange afforded the title compound

1

(58 mg, 75%): R_f) 0.40 (90:10 EtOAc/MeOH); H NMR (600

MHz, CD₃OD) δ 4.36 (d, J ) 3.0 Hz, 1H), 4.40 (dd, J ) 10.8, 3.0

Hz, 1H), 4.45-4.50 (m, 2H), 4.81 (t, J ) 5.4 Hz, 1H), 6.25 (d, J

) 5.4 Hz, 1H), 6.75-6.79 (m, 2H), 7.28 (t, J ) 7.8 Hz, 1H), 7.35

(t, J ) 7.8 Hz, 1H), 7.47 (t, J ) 7.8 Hz, 2H), 7.50-7.53 (m, 1H),

7.67 (d, J ) 7.8 Hz, 1H), 7.70 (d, J ) 7.8 Hz, 2H), 7.94 (d, J )

7.8 Hz, 1H), 8.38 (t, J ) 7.8 Hz, 1H), 8.49-8.51 (m, 1H), 8.63 (s,

1H); ¹³C NMR (150 MHz, CD₃OD) δ 70.0, 72.6, 76.1, 84.5, 89.5,

118.0, 119.2, 449.4, 120.7, 128.0, 128.3, 128.5, 128.6, 129.6,

129.96, 130.03, 131.6, 134.5, 140.4, 141.5, 142.59, 142.61, 152.4,

157.3, 161.1, 162.2, 175.5; HRMS (ESI-) calcd for C₂₉H₂₅N₆O₈S

[M - H]^-617.1460, found 617.1456 (error 0.6 ppm).

2-(Biphen-4-yl)-2′,3′-O-isopropylideneadenosine (81). To a solu-

tion of compound 75 (274 mg, 0.63 mmol, 1.0 equiv) in 1,4-dioxane

(10 mL) were added 4-biphenylboronic acid (251 mg, 1.27 mmol,

2.0 equiv), Pd(OAc)₂(28 mg, 0.126 mmol, 0.2 equiv), 2-(biphe-

nyl)dicyclohexylphosphine (66 mg, 0.19 mmol, 0.30 equiv), K₃PO₄

(334 mg, 1.58 mmol, 2.5 equiv), and the mixture was heated at

110 °C for 12 h. After cooling to room temperature, the mixture

was diluted with EtOAc (50 mL), ﬁltered, and concentrated.

Puriﬁcation by ﬂash chromatography (gradient from 80-100%

EtOAc/hexane) afforded the title compound (192 mg, 67%) as a

white solid: R_f) 0.20 (80:20 EtOAc/hexane); ¹H NMR (600 MHz,

DMSO-d₆) δ 1.37 (s, 3H), 1.59 (s, 3H), 3.53-3.57 (m, 1H),

3.59-3.62 (m, 1H), 4.21-4.24 (m, 1H), 5.03 (t, J ) 5.4 Hz, 1H),

5.11 (dd, J ) 6.0, 2.4 Hz, 1H), 5.52 (dd, J ) 6.0, 2.4 Hz, 1H),

6.26 (d, J ) 2.4 Hz, 1H), 7.37-7.42 (m, 3H), 7.50 (t, J ) 7.8 Hz,

2H), 7.75 (d, J ) 7.8 Hz, 2H), 7.79 (d, J ) 8.4 Hz, 2H), 8.37 (s,

1H), 8.44 (d, J ) 8.4 Hz, 2H); ¹³C NMR (150 MHz, DMSO-d₆) δ

25.3, 27.1, 61.6, 81.5, 83.4, 86.8, 89.1, 113.1, 118.2, 126.5, 126.7,

127.7, 128.3, 129.0, 137.3, 139.6, 140.4, 141.2, 149.9, 155.9, 157.7;

HRMS (APCI+) calcd for C₂₅H₂₆N₅O₄[M + H]⁺460.1979, found

460.2078 (error 21.5 ppm).

2-(Biphen-4-yl)-2′,3′-O-isopropylidene-5′-O-(sulfamoyl)adenos-

ine (88). Sulfamoyl chloride (66 mg, 0.57 mmol, 4.0 equiv) was

added to a solution of 81 (66 mg, 0.14 mmol, 1.0 equiv) in DMA

(1.0 mL) at 0 °C. The solution was stirred for 4 h at 0 °C and then

partitioned between EtOAc (25 mL) and H₂O (25 mL). The organic

layer was separated, washed with H₂O (3 × 25 mL), saturated

aqueous NaCl (25 mL), dried (Na₂SO₄), and concentrated to afford

the title compound (75 mg, 97%) as a white solid: R_f) 0.30 (80:

1

20 EtOAc/benzene); H NMR (600 MHz, DMSO-d₆) δ 1.38 (s,

V_i⁄V₀)

(1)

1 + ([I] ⁄ K_I^{app h}

)

3H), 1.60 (s, 3H), 4.16 (dd, J ) 10.8, 6.0 Hz, 1H), 4.28 (dd, J )

10.8, 5.4 Hz, 1H), 4.45 (td, J ) 5.7, 3.6 Hz, 1H), 5.20 (dd, J )

6.0, 3.6 Hz, 1H), 5.56 (dd, J ) 6.0, 2.4 Hz, 1H), 6.34 (d, J ) 2.4

Hz, 1H), 7.40 (t, J ) 7.8 Hz, 1H), 7.44 (br s, 2H), 7.50 (t, J ) 7.8

Hz, 2H), 7.58 (s, 2H), 7.75 (d, J ) 7.8 Hz, 2H), 7.80 (d, J ) 7.8

Hz, 2H), 8.34 (s, 1H), 8.43 (d, J ) 7.8 Hz, 2H); ¹³C NMR (150

MHz, DMSO-d₆) δ 25.2, 27.0, 68.1, 81.1, 83.3, 83.6, 88.8, 113.7,

118.2, 126.56, 126.64, 127.7, 128.3, 129.0, 137.2, 139.6, 140.3,

141.2, 149.8, 155.9, 157.9; HRMS (APCI+) calcd for C₂₅H₂₇N₆O₆S

[M + H]⁺539.1707, found 539.1711 (error 0.7 ppm).

([E] - [I] - K^a_I^pp) + ([E] - [I] - K^app)²+ 4[E][K^a_I^pp

]

√

I

V_i⁄ V₀)

2[E]

(2)

For assays involving FadD enzymes, the assay buffer and incubation

times were identical, but 0.30-3.0 µM corresponding FadD protein

was employed.

Isothermal Calorimetry. ITC displacement titration experiments

were performed with a Microcal VP-ITC titration microcalorimeter

(Microcal, Inc.) using salicylic acid as the weak ligand (K_D) 1.63

( 0.52 µM ⁵¹).¹⁰⁰MbtA was dialyzed (2 × 1 L) against 30 mM

Tris-HCl, pH 8.0, 1 mM MgCl₂, and 1 mM TCEP for 24 h prior to

titrations. MbtA concentration was determined by active site titration

as described²¹and diluted to 4.0 µM with dialysate buffer and

50-200 mM salicylic acid immediately prior to the experiment. A

solution of the ligand 26 was prepared in dialysate buffer containing

50-200 mM salicylic acid. Protein and ligand solutions were

degassed by vacuum aspiration (5-10 min) at room temperature

2-(Biphen-4-yl)-5′-O-[N-(2-hydroxybenzoyl)sulfamoyl]adenos-

ine Sodium Salt (29). Compound 88 (70 mg, 0.13 mmol, 1.0 equiv)

was coupled to compound 33 using the general procedure for

salicylation. The mixture was ﬁltered, washing with DMF (10 mL),

and the ﬁltrate was concentrated to provide the crude product as a

white solid. The crude product was dissolved in MeOH (10 mL)

and then slowly concentrated 3-fold by blowing down with a stream

of nitrogen gas to provide a white precipitate, which was collected

to yield 2-(biphen-4-yl)-2′,3′-O-isopropylidene-5′-O-{N-[(2-methoxy-

methoxy)benzoyl]sulfamoyl}adenosine cesium salt (67 mg, 73%):

5368 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Neres et al.

prior to loading the samples in the ITC cell and syringe. All

titrations were carried out at 25 °C with a stirring speed of 264

rpm and a 900 s duration between 10 µL injections. The initial

injection was not used for data ﬁtting. Titrations were run past the

point of enzyme saturation to determine the heat of dilution. The

heats of dilution were negligible in all cases and were subtracted

from the respective titrations prior to data analysis. Thermodynamic

parameters N (stoichiometry), apparent K_A^app(association constant),

and apparent ∆H^app(enthalpy change) were obtained by nonlinear

least-squares ﬁtting of the experimental data using a single-site-

binding model of the Origin software package (version 5.0) provided

with the instrument. The true K_Aand ∆H values were obtained

from the apparent values using eqs 3 and 4, where [Sal] represents

the concentration of salicylic acid and ∆H_(Sal)and K_A(Sal)correspond

to the enthalpy and association constant of salicylic acid with MbtA

(K_A(Sal)) 6.64 × 10⁵M^-1, ∆H_(Sal)) -6.77 kcal/mol).⁵¹

5′-O-(Sulfamoyl)adenosine (AMS, a known potent inhibitor of

protein synthesis³⁷) was used as a positive control.

Acknowledgment. This research was supported by a grant

from the NIH (Grant R01AI070219) and funding from the

Center for Drug Design, Academic Health Center, University

of Minnesota to C.C.A. We thank the Minnesota Supercom-

puting Institute for computing time. This research supported

in part by the Intramural Research Program of the NIH,

National Institute of Allergy and Infectious Disease.

Supporting Information Available: Table of HPLC purities

of 7-29, ¹H and ¹³C NMR data of all ﬁnal targets 7-29 and

intermediates 35-44, molecular dynamics simulations, QM/MM

studies, and complete author list for refs 58 and 88. This material

is available free of charge via the Internet at http://pubs.acs.org.

K_A

K^a_A^pp

)

(3)

References

1 + [Sal]K_A(Sal)

∆H^app+ ∆H_(Sal)[Sal]K_A(Sal)

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(4)

[1 + [Sal]K_A(Sal)

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(5)

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JM800567V

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Inhibition of siderophore biosynthesis in Mycobacterium tuberculosis with nucleoside bisubstrate analogues: Structure-activity relationships of the nucleobase domain of 5′-O-[N-(salicyl)sulfamoyl]adenosine

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